OLIGONUCLEOTIDES FOR SARS-CoV-2 MODULATION

ABSTRACT

This disclosure relates to novel SARS-CoV-2 targeting sequences. Novel SARS-CoV-2 targeting oligonucleotides for the treatment of SARS-CoV-2 infection are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 63/031,222, filed May 28, 2020, and U.S. Provisional Application Ser. No. 63/084,817, filed Sep. 29, 2020, the entire disclosures of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 8, 2021, is named 718622_UM9-258_ST25.txt and is 520,824 bytes in size.

FIELD OF THE INVENTION

This disclosure relates to novel SARS-CoV-2 targeting sequences, novel branched oligonucleotides, and novel methods for treating and preventing SARS-CoV-2-related infection.

BACKGROUND

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is a highly infectious virus that causes severe respiratory illness. The SARS-CoV-2 genome encodes for four structural proteins, S (spike), E (envelope), M (membrane) and N (nucleocapsid). The spike protein plays a critical role in viral entry into a host cell. SARS-CoV-2 is the causative agent of the COVID-19 epidemic that has infected and kills millions of people worldwide.

RNAi-based therapeutics are revolutionizing human medicine. Currently, a single subcutaneous injection of chemically modified oligonucleotide compounds supports up to 12-months of target silencing in the liver with a clean adverse events profile. The ability to develop RNAi-based drugs is dependent on efficient delivery to the targeted tissues. Currently, the liver is the only tissue validated for clinical delivery.

With the current clinical approaches, it is not possible to halt or cure SARS-CoV-2 infection. The highly infectious virus is spreading throughout the world, leaving a path of destruction and death. Survivors of a severe infection with SARS-CoV-2 often present with long lasting lung injury and scarring.

There is a clear need for a therapeutic that can effectively neutralize SARS-CoV-2 particles from causing infection, and especially to selectively do so in the lung. This could be accomplished using optimized RNAi-based therapeutics, which is addressed in the present application.

SUMMARY

In one aspect, the disclosure provides an RNA molecule having a length of from about 8 nucleotides to about 80 nucleotides; and a nucleic acid sequence that is substantially complementary to a SARS-CoV-2 nucleic acid sequence of SEQ ID NO: 1. In certain embodiments, the RNA molecule is from 8 nucleotides to 80 nucleotides in length (e.g., 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, 50 nucleotides, 51 nucleotides, 52 nucleotides, 53 nucleotides, 54 nucleotides, 55 nucleotides, 56 nucleotides, 57 nucleotides, 58 nucleotides, 59 nucleotides, 60 nucleotides, 61 nucleotides, 62 nucleotides, 63 nucleotides, 64 nucleotides, 65 nucleotides, 66 nucleotides, 67 nucleotides, 68 nucleotides, 69 nucleotides, 70 nucleotides, 71 nucleotides, 72 nucleotides, 73 nucleotides, 74 nucleotides, 75 nucleotides, 76 nucleotides, 77 nucleotides, 78 nucleotides, 79 nucleotides, or 80 nucleotides in length).

In certain embodiments, the RNA molecule is from 10 to 50 nucleotides in length (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, 30 nucleotides, 31 nucleotides, 32 nucleotides, 33 nucleotides, 34 nucleotides, 35 nucleotides, 36 nucleotides, 37 nucleotides, 38 nucleotides, 39 nucleotides, 40 nucleotides, 41 nucleotides, 42 nucleotides, 43 nucleotides, 44 nucleotides, 45 nucleotides, 46 nucleotides, 47 nucleotides, 48 nucleotides, 49 nucleotides, or 50 nucleotides in length).

In certain embodiments, the RNA molecule comprises about 15 nucleotides to about 25 nucleotides in length. In certain embodiments, the RNA molecule is from 15 to 25 nucleotides in length (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides in length).

In one aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of SEQ ID NO: 1.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 2-10.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide targets in Table 6A.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions of 7a_27751, N_29293, Orfla_2290, and Orflab_18571 in Table 6A.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide targets of 7a_27751, N_29293, Orfla_2290, and Orflab_18571 in Table 6A.

In certain embodiments, the oligonucleotide compound comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the oligonucleotide compound comprises no more than 3 mismatches with the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. In certain embodiments, the oligonucleotide compound comprises full complementarity to the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10.

In another aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to an Angiotensin I Converting Enzyme 2 (ACE2) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 1 ID or Table 12C.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to an ACE2 nucleic acid sequence of any one of the 45-nucleotide target gene regions of ACE2_119, ACE2_336, ACE2_349, ACE_1034, ACE_1775, ACE_784, ACE_908, and ACE_1071, as recited in Table 12C

In another aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a FURIN nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 1 IC or Table 12D.

In certain embodiments, the oligonucleotide compound comprises a sequence substantially complementary to a FURIN nucleic acid sequence of any one of the 45-nucleotide target gene regions of FURIN_443, FURIN_1959, FURIN_2711, FURIN_2712, FURIN_3524, and FURIN_3526, as recited in Table 12D.

In one aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to an Interleukin 6 (IL-6) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 11B or Table 12B.

In one aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to an Interleukin 6 Receptor (IL-6R) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 12E.

In one aspect, the disclosure provides an oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a Transmembrane Serine Protease 2 (TMPRSS2) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 11A or Table 12A.

In certain embodiments, the oligonucleotide compound comprises one or more naturally occurring nucleotides.

In certain embodiments, the oligonucleotide compound comprises one or more modified nucleotide.

In certain embodiments, the one or more modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.

In certain embodiments, each modification of the ribose group is independently selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, and a constrained nucleotide.

In certain embodiments, the constrained nucleotide is selected from the group consisting of a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^(NC)), an alpha-L-locked nucleic acid, a tricyclo-DNA, and any combination thereof.

In certain embodiments, the constrained nucleotide is a locked nucleic acid (LNA), a 2′-(S)-constrained ethyl (S-cEt) nucleotide, and a combination thereof

In certain embodiments, each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.

In certain embodiments, each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification.

In certain embodiments, the modification of the phosphate group is phosphorothioate

In certain embodiments, the oligonucleotide compound comprises 4-16 phosphorothioate modifications. In certain embodiments, the oligonucleotide compound comprises 6-13 phosphorothioate modifications.

In certain embodiments, the oligonucleotide compound comprises at least one modified internucleotide linkage.

In certain embodiments, the oligonucleotide compound comprises at least one modified internucleotide linkage of Formula I:

wherein: B is a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; X is selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, OR, NH—, NH₂, S—, and SH; Z is selected from the group consisting of O and CH₂; R is a protecting group; and

is an optional double bond.

In certain embodiments, the oligonucleotide compound comprises at least 80% chemically modified nucleotides. In certain embodiments, the oligonucleotide compound is fully chemically modified.

In certain embodiments, the oligonucleotide compound comprises an antisense oligonucleotide or a double stranded (ds) RNA.

In certain embodiments, the dsRNA comprises an antisense strand and a sense strand. In certain embodiments, the antisense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the sense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments, the dsRNA comprises a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

In certain embodiments, the dsRNA comprises a blunt-end. In certain embodiments, the dsRNA comprises at least one single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang. In certain embodiments, the dsRNA comprises a 2-nucleotide single stranded nucleotide overhang or a 5-nucleotide single stranded nucleotide overhang.

In certain embodiments, the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications.

In certain embodiments, the antisense strand comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises about 70% to 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises 100% 2′-O-methyl nucleotide modifications.

In certain embodiments, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of sense strand. In certain embodiments, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of the sense strand.

In certain embodiments, the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate. In certain embodiments, the antisense strand comprises a 5′ vinyl phosphonate.

In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In certain embodiments, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not a 2′-methoxy-ribonucleotide; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 6, 7, 8 and 10 from the 5′ end of the sense strand are not a 2′-methoxy-ribonucleotide; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In certain embodiments, the dsRNA further comprises 1 to 5 internucleotide linkages of Formula I. In certain embodiments, the 3′ end of the antisense strand comprises 1 to 5 internucleotide linkages of Formula I. In certain embodiments, the 3′ end of the antisense strand comprises 4 consecutive internucleotide linkages of Formula I.

In certain embodiments, a functional moiety is linked to one or both of the 5′ end and 3′ end of the antisense strand. In certain embodiments, a functional moiety is linked to one or both of the 5′ end and 3′ end of the sense strand. In certain embodiments, a functional moiety is linked to the 3′ end of the sense strand.

In certain embodiments, the functional moiety comprises a hydrophobic moiety. In certain embodiments, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof. In certain embodiments, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). In certain embodiments, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In certain embodiments, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In certain embodiments, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

In certain embodiments, the functional moiety is linked to one or both of the antisense strand and sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the divalent or trivalent linker is selected from the group consisting of

wherein n is 1, 2, 3, 4, or 5.

In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

In certain embodiments, the linker comprises a dTdT dinucleotide.

In certain embodiments, the functional moiety is linked to the 3′ end of the sense strand by a dTdT dinucleotide followed by the linker

wherein n is 1.

In certain embodiments, the trivalent linker further links a phosphodiester or phosphodiester derivative.

In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of

wherein X is O, S or BH₃.

In certain embodiments, the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages.

In one aspect, the disclosure provides a double stranded (ds) RNA, comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not a 2′-methoxy-ribonucleotide; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 6, 7, 8 and 10 from the 5′ end of the sense strand are not a 2′-methoxy-ribonucleotide; (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; and (9) the antisense strand comprises at least one modified intersubunit linkages of Formula II:

wherein: B is a base pairing moiety; W is O or O(CH₂)_(n) ¹, wherein n¹ is 1 to 10; X is selected from the group consisting of H, OH, OR¹, F, SH, SR, NR² ₂ and C₁₋₆-alkoxy; Y is selected from the group consisting of O—, OH, OR, OR², NH—, NH₂, NR² ₂, BH₃, S—, R¹, and SH; Z is O or O(CH₂)_(n) ² wherein n² is 1 to 10; R¹ is alkyl, allyl or aryl; and R² is alkyl, allyl or aryl.

In certain embodiments, the 3′ end of the antisense strand comprises four consecutive modified intersubunit linkages of Formula II.

In certain embodiments, the antisense strand comprises a 5′ vinyl phosphonate.

In certain embodiments, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

In certain embodiments, a functional moiety is linked to the 3′ end of the sense strand.

In certain embodiments, the functional moiety comprises Eicosapentaenoic acid (EPA) or Docosanoic acid (DCA).

In certain embodiments, a functional moiety is linked to the 3′ end of the sense strand by a linker.

In certain embodiments, the linker comprises a dTdT dinucleotide.

In certain embodiments, the functional moiety is linked to the 3′ end of the sense strand by a dTdT dinucleotide followed by the linker

wherein n is 1.

In one aspect, the disclosure provides a combination comprising two or more oligonucleotide compounds or dsRNA recited above, wherein each oligonucleotide compound or dsRNA in the combination comprises complementarity to a different SARS-CoV-2 nucleic acid sequence.

In certain embodiments, the combination comprises two, three, four, or five oligonucleotide compounds or dsRNA.

In one aspect, the disclosure provides a combination comprising two or more oligonucleotide compounds for inhibiting the expression of a SARS-CoV-2 gene in a cell of an organism, wherein each oligonucleotide compound in the combination comprises complementarity to a different SARS-CoV-2 nucleic acid sequence.

In certain embodiments, the combination comprises a first oligonucleotide compound, a second oligonucleotide compound, and a third oligonucleotide compound, wherein:

i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_8744, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_8744, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region N_29293, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region N_29293, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26470, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27123, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27032, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26369, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27032, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 3a_25868, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23774, as recited in Table 6A.

In one aspect, the disclosure provides a combination comprising one or more oligonucleotide compounds for inhibiting the expression of a SARS-CoV-2 gene and one or more oligonucleotide compounds for inhibiting the expression of one or more of an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and a IL-6R gene.

In one aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of one or more of a SARS-CoV-2 gene, an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and a IL-6R gene in a cell of an organism, comprising the oligonucleotide compound, dsRNA, or combination recited above and a pharmaceutically acceptable carrier.

In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more of the SARS-CoV-2 genes, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 50%. In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more of the SARS-CoV-2 genes, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 75%.

In one aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of one or more SARS-CoV-2 genes in a cell of an organism, comprising the oligonucleotide compound, dsRNA, or combination recited above and a pharmaceutically acceptable carrier.

In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more SARS-CoV-2 genes by at least 50%. In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more SARS-CoV-2 genes by at least 75%.

In one aspect, the disclosure provides a method for inhibiting expression of a SARS-CoV-2 gene in a cell of an organism, the method comprising: (a) introducing into the cell an oligonucleotide compound, dsRNA, or combination recited above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SARS-CoV-2 gene, thereby inhibiting expression of the SARS-CoV-2 gene in the cell.

In one aspect, the disclosure provides a method for inhibiting expression of one or more of a SARS-CoV-2 gene, an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and an IL-6R gene in a cell, the method comprising: (a) introducing into the cell an oligonucleotide compound, dsRNA, or combination recited above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene thereby inhibiting expression of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene in the cell.

In one aspect, the disclosure provides a method of treating or managing a SARS-CoV-2 infection, comprising administering to a patient in need of such treatment a therapeutically effective amount of the oligonucleotide compound, dsRNA, or combination recited above.

In certain embodiments, the oligonucleotide compound, dsRNA, or combination is administered by intratracheal (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

In certain embodiments, the oligonucleotide compound, dsRNA, or combination is administered sequentially or simultaneously.

In certain embodiments, administering the oligonucleotide compound, dsRNA, or combination causes a decrease in one or more of SARS-CoV-2 gene mRNA, ACE2 gene mRNA, FURIN gene mRNA, IL-6 gene mRNA, TMPRSS2 gene mRNA, and IL-6R gene mRNA in the lung.

In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 50%. In certain embodiments, the oligonucleotide compound, dsRNA, or combination inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 75%.

In one aspect, the disclosure provides a vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an oligonucleotide compound recited above.

In certain embodiments of the vector, the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 30%. In certain embodiments of the vector, the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 50%. In certain embodiments of the vector, the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 75%.

In one aspect, the disclosure provides a cell comprising the vector recited above.

In one aspect, the disclosure provides a recombinant adeno-associated virus (rAAV) comprising the vector recited above and an AAV capsid.

In one aspect, the disclosure provides a branched oligonucleotide compound comprising two or more of the oligonucleotide compounds or dsRNA recited above covalently bound to one another.

In certain embodiments, the oligonucleotide compounds are covalently bound to one another by way of a linker, spacer, a branching point, or a mixture thereof.

In one aspect, the disclosure provides a method of treating or managing a SARS-CoV-2 infection, comprising administering to a patient in need of such treatment a therapeutically effective amount of the branched oligonucleotide compound recited above.

In certain embodiments, the branched oligonucleotide compound is administered by intratracheal (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

In certain embodiments, the branched oligonucleotide compound accumulates in lung tissue to a greater extent than a non-branched oligonucleotide compound when administered by intratracheal (IT) injection.

In one aspect, the disclosure provides a branched RNA compound comprising: two or more RNA molecules comprising 15 to 35 nucleotides in length, and a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence, wherein the two or more RNA molecules are connected to one another by one or more moieties independently selected from a linker, a spacer and a branching point.

In certain embodiments, the branched RNA compound comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A.

In certain embodiments of the branched RNA compound, each RNA molecule comprises 15 to 25 nucleotides in length.

In certain embodiments of the branched RNA compound, each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, wherein each antisense strand independently comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A.

In certain embodiments, the branched RNA compound comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A. In certain embodiments of the branched RNA compound, each RNA molecule comprises no more than 3 mismatches with a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A. In certain embodiments, the branched RNA compound comprises full complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A.

In certain embodiments of the branched RNA compound, the antisense strand comprises a portion having the nucleic acid sequence of any one of the antisense strands recited in Table 6B.

In certain embodiments of the branched RNA compound, the antisense strand and/or sense strand comprises about 15 nucleotides to 25 nucleotides in length. In certain embodiments of the branched RNA compound, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments of the branched RNA compound, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

In certain embodiments of the branched RNA compound, the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs. In certain embodiments of the branched RNA compound, the dsRNA comprises a double-stranded region of 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs.

In certain embodiments of the branched RNA compound, the dsRNA comprises a blunt-end. In certain embodiments of the branched RNA compound, the dsRNA comprises at least one single stranded nucleotide overhang. In certain embodiments of the branched RNA compound, the dsRNA comprises between a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang.

In certain embodiments of the branched RNA compound, the dsRNA comprises naturally occurring nucleotides.

In certain embodiments of the branched RNA compound, the dsRNA comprises at least one modified nucleotide.

In certain embodiments of the branched RNA compound, the modified nucleotide comprises a 2′-O-methyl modified nucleotide, a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, or a non-natural base comprising nucleotide.

In certain embodiments of the branched RNA compound, the dsRNA comprises at least one modified internucleotide linkage. In certain embodiments of the branched RNA compound, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the branched RNA compound comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the branched RNA compound comprises 6-13 phosphorothioate internucleotide linkages.

In certain embodiments of the branched RNA compound, the dsRNA comprises at least one modified internucleotide linkage of Formula I:

wherein: B is a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; X is selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, OR, NH—, NH₂, S—, and SH; Z is selected from the group consisting of O and CH₂; R is a protecting group; and

is an optional double bond.

In certain embodiments of the branched RNA compound, the dsRNA comprises at least 80% chemically modified nucleotides. In certain embodiments of the branched RNA compound, the dsRNA is fully chemically modified. In certain embodiments of the branched RNA compound, the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments of the branched RNA compound, the antisense strand comprises at least 70% 2′-O-methyl nucleotide modifications. In certain embodiments of the branched RNA compound, the antisense strand comprises about 70% to 90% 2′-O-methyl nucleotide modifications. In certain embodiments of the branched RNA compound, the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications. In certain embodiments of the branched RNA compound, the sense strand comprises 100% 2′-O-methyl nucleotide modifications.

In certain embodiments of the branched RNA compound, the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand. In certain embodiments of the branched RNA compound, the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of sense strand. In certain embodiments of the branched RNA compound, the nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of the sense strand.

In certain embodiments of the branched RNA compound, the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, a 5′ alkenyl phosphonate, or a mixture thereof. In certain embodiments of the branched RNA compound, the antisense strand comprises a 5′ vinyl phosphonate.

In certain embodiments of the branched RNA compound, the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In certain embodiments of the branched RNA compound, the antisense strand comprises a 5′ vinyl phosphonate.

In certain embodiments of the branched RNA compound, the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length. In certain embodiments of the branched RNA compound, the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length.

In certain embodiments of the branched RNA compound, a functional moiety is linked to one or both of the 5′ end and 3′ end of the antisense strand. In certain embodiments of the branched RNA compound, a functional moiety is linked to one or both of the 5′ end and 3′ end of the sense strand. In certain embodiments of the branched RNA compound, a functional moiety is linked to the 3′ end of the sense strand.

In certain embodiments of the branched RNA compound, the functional moiety comprises a hydrophobic moiety. In certain embodiments of the branched RNA compound, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof. In certain embodiments of the branched RNA compound, the steroid is selected from the group consisting of cholesterol and Lithocholic acid (LCA). In certain embodiments of the branched RNA compound, the fatty acid is selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In certain embodiments of the branched RNA compound, the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, derivatives thereof, and metabolites thereof. In certain embodiments of the branched RNA compound, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

In certain embodiments of the branched RNA compound, the functional moiety is linked to one or both of the antisense strand and sense strand by a linker. In certain embodiments of the branched RNA compound, the linker comprises a divalent or trivalent linker.

In certain embodiments of the branched RNA compound, the divalent or trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4, or 5.

In certain embodiments of the branched RNA compound, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

In certain embodiments of the branched RNA compound, the trivalent linker further links a phosphodiester or phosphodiester derivative. In certain embodiments of the branched RNA compound, the phosphodiester or phosphodiester derivative is selected from the group consisting of:

wherein X is O, S or BH₃.

In certain embodiments of the branched RNA compound, the nucleotides at positions 1 and 2 from the 3′ end of sense strand, and the nucleotides at positions 1 and 2 from the 5′ end of antisense strand, are connected to adjacent ribonucleotides via phosphorothioate linkages.

In one aspect, the disclosure provides compound of formula (I):

L-(N)_(n)  (I)

wherein: L comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof, and wherein formula (I) optionally further comprises one or more branch point B, and one or more spacer S, wherein B is independently for each occurrence a polyvalent organic species or derivative thereof; S comprises independently for each occurrence an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or a combination thereof; and N is a double stranded nucleic acid comprising 15 to 35 bases in length comprising a sense strand and an antisense strand; wherein the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; the sense strand and antisense strand each independently comprise one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.

In certain embodiments, the compound has a structure selected from formulas (I-1)-(I-9):

In certain embodiments, the antisense strand comprises a 5′ terminal group R selected from the group consisting of:

In certain embodiments, the compound comprises the structure of formula (II):

wherein: X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

In certain embodiments, the compound comprises structure of formula (IV):

wherein: X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

In certain embodiments, L is structure L1:

In certain embodiments, R is R³ and n is 2.

In certain embodiments, L is structure L2:

In certain embodiments, R is R³ and n is 2.

In one aspect, the disclosure provides a delivery system for therapeutic nucleic acids having the structure of Formula (VI):

L-(cNA)_(n)  (VI)

wherein: L comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof, wherein formula (VI) optionally further comprises one or more branch point B, and one or more spacer S, wherein B comprises independently for each occurrence a polyvalent organic species or a derivative thereof; S comprises independently for each occurrence an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, or combinations thereof; each cNA, independently, is a carrier nucleic acid comprising one or more chemical modifications; each cNA, independently, comprises at least 15 contiguous nucleotides of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; and n is 2, 3, 4, 5, 6, 7 or 8.

In certain embodiments, the delivery system has a structure selected from formulas (VI-1)-(VI-9):

In certain embodiments, each cNA independently comprises a chemically-modified nucleotide.

In certain embodiments, the delivery system further comprises n therapeutic nucleic acids (NA), wherein each NA is hybridized to at least one cNA.

In certain embodiments, each NA independently comprises at least 16 contiguous nucleotides. In certain embodiments, each NA independently comprises 16-20 contiguous nucleotides.

In certain embodiments, each NA comprises an unpaired overhang of at least 2 nucleotides. In certain embodiments, the nucleotides of the overhang are connected via phosphorothioate linkages.

In certain embodiments, each NA, independently, is selected from the group consisting of DNAs, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, and guide RNAs.

In certain embodiments, each NA is substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A.

In one aspect, the disclosure provides a pharmaceutical composition for inhibiting the expression of a SARS-CoV-2 gene in an organism, comprising a compound or a system recited above, and a pharmaceutically acceptable carrier.

In certain embodiments, the compound or system inhibits the expression of the SARS-CoV-2 gene by at least 50%. In certain embodiments, the compound or system inhibits the expression of the SARS-CoV-2 gene by at least 75%.

In one aspect, the disclosure provides a method for inhibiting expression of a SARS-CoV-2 gene in a cell, the method comprising: (a) introducing into the cell a compound or a system recited above; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SARS-CoV-2 gene, thereby inhibiting expression of the SARS-CoV-2 gene in the cell.

In one aspect, the disclosure provides a method of treating or managing a SARS-CoV-2 infection comprising administering to a patient in need of such treatment or management a therapeutically effective amount of a compound or a system recited above.

In certain embodiments, the compound or system is administered to the lung of the patient.

In certain embodiments, the compound or system is administered by intratracheal (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof.

In certain embodiments, administering the compound or system causes a decrease in SARS-CoV-2 gene mRNA in one or more of the club cells and alveoli cells of the lung.

In certain embodiments, the compound or system inhibits the expression of the SARS-CoV-2 gene by at least 50%. In certain embodiments, the compound or system inhibits the expression of the SARS-CoV-2 gene by at least 75%.

In certain embodiments, the compound or system accumulates in lung tissue to a greater extent than a non-branched oligonucleotide compound when administered by intratracheal (IT) injection.

In one aspect, the disclosure provides a method of delivering an oligonucleotide compound to the lung of a patient, comprising administering the oligonucleotide compound, wherein the oligonucleotide compound is conjugated to a functional moiety selected from Eicosapentaenoic acid (EPA) and Docosanoic acid (DCA).

In certain embodiments, the oligonucleotide compound is a dsRNA comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end. In certain embodiments, the dsRNA is conjugated to the sense strand 3′ end.

In certain embodiments, the functional moiety is conjugated to the sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker.

In certain embodiments, the divalent or trivalent linker is selected from the group consisting of:

wherein n is 1, 2, 3, 4, or 5.

In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof.

In certain embodiments, the trivalent linker further links a phosphodiester or phosphodiester derivative.

In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of:

wherein X is O, S or BH₃.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings.

FIG. 1A-1B depict an exemplary chemically modified siRNA (FIG. 1A), and a schematic of the SARS-CoV-2 genome (FIG. 1B).

FIG. 2 depicts a diagram of siRNA and ASO target positions on encoded proteins in the SARS-CoV-2 Genome. siRNAs were designed to target nine genes encoding SARS-CoV-2 proteins: orfla, orflab, spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), nucleocapsid protein (N), and accessory proteins 3a, 8b, 7a. Grey arrows indicate siRNA and ASO target positions. Inset shows detailed view of siRNA target positions on genes in the 3′ region of the genome.

FIG. 3A-3B depict an alignment of siRNAs and ASOs selected for synthesis directed to six closely-related CoVs using a novel algorithm. Aligned genome regions of CoVs are shaded based on homology with darker coloring indicating higher homology with respect to SARS-CoV-2. The siRNA position is indicated on the top. Per position percent homology of SARS-CoV-2 to the six related CoVs is plotted on the bottom. SiRNA with low homology scores of 59 are shown in FIG. 3A (SEQ ID NOS 2583-2589, respectively, in order of appearance). SiRNA with a high homology score are shown in FIG. 3B (SEQ ID NOS 2590-2596, respectively, in order of appearance). Gaps in alignment are indicated with dashes (-).

FIG. 4A-4B depict siRNA and ASO target selection based on the ability to target many SARS-CoV-2 genomes from patient isolates. FIG. 4A, siRNAs and ASOs were selected to target regions of the 9 selection genes with low mutation rates in other coronaviruses. FIG. 4B, the proportion of SARS-CoV-2 variants from patient isolates targeted by all selected siRNAs. FIG. FIG. 5A-5I depicts the identification of siRNA hits for SARS-CoV-2. SiRNAs targeting different genes in the SARS-CoV-2 genome were tested for silencing efficacy.

FIG. 5A, gene orfla, FIG. 5B, gene 3a; FIG. 5C, gene 7a, FIG. 5D, gene orflab, FIG. 5E, gene E, FIG. 5F, gene 8b, FIG. 5G, gene S, FIG. 5H, gene M, FIG. 5I, gene N. SiRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: 1.5 uM; Time point: 72 hours.

FIG. 6A-6I depict the identification of ASO hits for SARS-CoV-2. ASOs targeting different genes in the SARS-CoV2 genome were tested for silencing efficacy. FIG. 6A, gene orfla, FIG. 6B, gene Orflab; FIG. 6C, gene S, FIG. 6D, gene 3a, FIG. 6E, gene E, FIG. 6F, gene M, FIG. 6G, gene 7a, FIG. 6H, gene 8b, FIG. 6I, gene N. ASOs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: 1.5 uM; Time point: 72 hours.

FIG. 7A-7B depict the identification of siRNA hits for SARS-CoV2 and mapping onto genes in the SARS-CoV-2 genome. FIG. 7A, SARS-CoV-2 genome. FIG. 7B, siRNAs targeting different genes in the SARS-CoV2 genome tested for silencing efficacy. siRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: 1.5 uM; Time point: 72 hours.

FIG. 8A-8B depicts the identification of ASO hits for SARS-CoV-2 and mapping onto genes in the SARS-CoV-2 genome. FIG. 8A, SARS-CoV-2 genome. FIG. 8B, LNA gapmers targeting different genes in the SARS-CoV-2 genome were tested for silencing efficacy. ASOs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: 1.5 μM; Time point: 72 hours.

FIG. 9A-9I depict the validation and determination of IC₅₀ values. SiRNAs targeting different genes in the SARS-CoV-2 genome were tested for silencing efficacy in 8-point dose response studies. FIG. 9A, gene orfla, FIG. 9B, gene Orflab; FIG. 9C, gene Spike, FIG. 9D, gene 3a, FIG. 9E, gene Envelope, FIG. 9F, gene Membrane, FIG. 9G, gene Orf7a, FIG. 9H, gene Orf8a, FIG. 9I, gene Nucleocapsid. siRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: Top=1.5 μM; Time point: 72 hours.

FIG. 10 depicts a schematic showing SARS-CoV-2 genes and their functions. Non-structural genes undergo primary translation while structural and accessory proteins are translated from sub-genomic mRNAs.

FIG. 11A-11E depict validation and determination of IC₅₀ values for siRNA cocktails targeting SARS-CoV-2 Genes. FIG. 11A, replication cocktails; FIG. 11B, Replication/immuno cocktails; FIG. 11C, Replication/Capsid cocktails; FIG. 11D, Immuno/Capsid cocktails; and FIG. 11E, Replication/Immuno/Capsid cocktails. siRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: Top=1.5 uM; Time point: 72 hours.

FIG. 12A-12B depict design of siRNAs targeting ACE2 (FIG. 12A) and FURIN (FIG. 12B).

FIG. 13A-13B depict the identification of siRNA hits for ACE2 (FIG. 13A) and FURIN (FIG. 13B). siRNAs were tested in human Hacat cells and silencing was assessed using the QuantiGene assay and confirmed using psicheck reporter system. Concentration: 1.5 μM; Time point: 72 hours.

FIG. 14A-14B depict validation and determination of IC₅₀ values for siRNAs targeting ACE2 (FIG. 14A) and FURIN (FIG. 14B). siRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system. Concentration: Top=1.5 μM; Time point: 72 hours.

FIG. 15A-15D depict validation and determination of IC₅₀ values for siRNAs targeting FURIN. FIG. 15A, 2711; FIG. 15B, 2712; FIG. 15C, 3524; FIG. 15D, 3526. siRNAs were tested in HaCat cells and silencing was assessed using QuantiGene. Concentration: Top=1.5 μM; Time point: 72 hours.

FIG. 16A-16B depict the identification of ASO hits for ACE2 and FURIN. Twelve LNA gapmers targeting ACE2 (FIG. 16A) and FURIN (FIG. 16B) were tested for silencing efficacy. ASOs were tested in human U20S cells and silencing was assessed using QRT-PCR assay. Concentration: 1.5 μM; Time point: 72 hours.

FIG. 17A-17B depict the identification of ASO hits for ACE2 and FURIN. Twelve LNA gapmers targeting ACE2 (FIG. 17A) and FURIN (FIG. 17B), were tested for silencing efficacy. ASOs were tested in human U20S cells and silencing was assessed using QRT-PCR assay. Concentration: 1.5 μM; Time point: 72 hours.

FIG. 18A-18B depict validation and determination of IC₅₀ values for ASOs targeting ACE2 (FIG. 18A) and FURIN (FIG. 18B). Concentration: Top=1.5 μM; Time point: 5 days.

FIG. 19A-19F show that the presence of a two-thymidine linker between the conjugate and the siRNA does not impact siRNA tissue distribution profile. The siRNA structural configurations studied to evaluate the impact of the nature of the linker on distribution is shown in FIG. 19A, FIG. 19C, and FIG. 19E. The corresponding bar graphs for siRNA accumulation in the various tissues are shown in FIG. 19B, FIG. 19D, and FIG. 19F, respectively. Shown are siRNA strand accumulation of DCA-conjugated siRNA in liver, kidney, spleen, lung, heart, muscle and fat 1-week after a single SC injection with 20 mg/kg (n=5-6 mice per group ±SD), measured by PNA hybridization assay.

FIG. 20A-20B show that the presence of a two-thymidine linker increases DCA-conjugated siRNA silencing in multiple tissues. FIG. 20A, effect on huntingtin mRNA expression. FIG. 20B, effect on cyclophilin B mRNA expression. The presence of a two-thymidine linker increases DCA-conjugated siRNA silencing in multiple tissues. SC injection (FVB/N mice); 20 mg/kg; collection of tissues one week after injection; n=6 per group. Huntingtin (Htt) (A.) or Cyclophilin B (Ppib) (B.) mRNA levels were measured using QuantiGene® (Affymetrix), normalized to a housekeeping gene, Hprt (Hypoxanthine-guanine phosphoribosyl transferase), and presented as percent of PBS (Phosphate buffered saline) control (mean±SD). Data analysis: t test (****P<0.0001, ***P<0.001, **P<0.01, *P<0.1).

FIG. 21 depicts designs and structural configurations of siRNAs for lung delivery via systemic (SC). Schematic of siRNA structural configuration studied to evaluate the impact of the chemical composition on siRNA distribution and efficacy.

FIG. 22 depicts distribution and accumulation of siRNAs conjugated to DCA and containing different numbers of 3′ exNA modifications and phosphorothioates. These data show increased accumulation of DCA-conjugated siRNAs with exNA modifications compared to those without exNAs in all tissues including the lungs. SC, 20 mg/kg, n=3, 1 week, PNA hybridization assay. p2 scaffold: 4PS-exNa≈7PS in liver, spleen, lung; 4PS-exNa>7 PS in heart, muscle, fat; 7PS>4PS-exNa in kidney. p5 scaffold: 4PS-exNa>2PO-exNa 2PS-exNa≥4 PS>2PS.

FIG. 23A-23H depict target mRNA silencing (Htt) after systemic administration of siRNAs conjugated to DCA and containing different numbers of 3′ exNA modifications and phosphorothioates in various organ tissues. FIG. 23A, liver; FIG. 23B, kidney; FIG. 23C, spleen; FIG. 23D, muscle; FIG. 23E, lung; FIG. 23F, heart; FIG. 23G, adrenal glands; FIG. 23H, Fat. These data show increased silencing of DCA-conjugated siRNAs with exNA modifications compared to those without exNAs in all tissues including the lungs. SC, 20 mg/kg, n=5, 1 week, bDNA QuantiGene assay.

FIG. 24 depicts designs of siRNAs for lung delivery via intratracheal administration. Schematic of siRNA structural configuration studied to evaluate the impact of the chemical composition on siRNA distribution and efficacy.

FIG. 25A-25B depict siRNA accumulation after intratracheal administration. FIG. 25A, Distribution and delivery throughout the lung of mono and divalent siRNAs (Cy-3) compared to PBS controls; FIG. 25B, distribution and delivery throughout the lung of EPA and DCA conjugated siRNAs (Cy-3) compared to PBS controls. Intratracheal; 20 nmol for mono, 40 nmol for di; n=2, 24 h, 5×, Scale=1 mm. Subcutaneous; 40 nmol; n=3, 48 h, 5×, Scale=1 mm.

FIG. 26A-26C depict mono and di-valent siRNA accumulation after intratracheal administration. FIG. 26A, distribution and delivery throughout the lung of mono and divalent siRNAs (Cy-3) compared to PBS controls; FIG. 26B, distribution and delivery throughout the lung of mono and divalent siRNAs (Red, Cy3) to club cells (epithelial) (green) of the lungs compared to PBS controls; FIG. 26C, distribution and delivery throughout the lung of mono and divalent siRNAs (Red, Cy3) to alveoli type II cells (green) in the lung compared to PBS controls. Divalent siRNAs distribute to all cells of the lungs and saturate both alveolar and epithelial (club) cells 24 hours after intratracheal administration.

FIG. 27A-27C depict EPA and DCA conjugated siRNA distribution throughout the lungs after subcutaneous (SC) administration). FIG. 27A, distribution and delivery throughout the lung of EPA and DCA conjugated siRNAs (Cy-3) compared to PBS controls; FIG. 27B, distribution of EPA and DCA conjugated siRNAs (Red, Cy3) to club cells (epithelial) (green) of the lungs compared to PBS controls; FIG. 27C, distribution of EPA and DCA conjugated siRNAs (Red, Cy3) to alveoli cells (green) in the lung compared to PBS controls.

FIG. 28A-28D depict quantification of siRNA accumulation after systemic and intratracheal administration. FIG. 28A, fluorescence uptake in alveolar cells; FIG. 28B, percent in alveoli cells/total cells; FIG. 28C, fluorescence uptake in club cells; FIG. 28D, percent in club cells/total cells.

FIG. 29A-29G depict quantification of EPA and DCA conjugated siRNA accumulation after systemic administration. FIG. 29A, distribution in various lung cells; FIG. 29B, distribution by cell type; FIG. 29C-29G, CY3 signals in total cells, immune cells, endothelial cells, epithelial cells and fibroblasts, respectively. Using flow cytometry siRNA accumulation was quantified after systemic (SC) administration of EPA and DCA conjugated siRNAs.

FIG. 30A-30G depict quantification of siRNA accumulation after intratracheal administration. Using flow cytometry, siRNA accumulation was quantified after intratracheal administration of mono and di-valent siRNAs. FIG. 30A, distribution in various lung cells;

FIG. 30B, distribution by cell type; FIG. 30C-30G, CY3 signals in total cells, immune cells, endothelial cells, epithelial cells and fibroblasts, respectively. Divalent siRNAs showed the highest amount of uptake in all cell types compared to other siRNAs.

FIG. 31A-31B depict distribution and accumulation of mono and di-siRNAs. FIG. 31A, di-siRNAs after intratracheal administration; FIG. 31B, di-siRNAs after intratracheal administration and DAC/EPA siRNA after SC injection. Intratracheal or SC, 7.5 and 15 nmol and 40 nmol, n=3, 1 week, PNA hybridization assay.

FIG. 32A-32H show target mRNA silencing (Htt) after intratracheal administration of mono and di-siRNAs in various tissues. FIG. 32A, liver; FIG. 32B, kidney;

FIG. 32C, spleen; FIG. 32D, lung; FIG. 32E, heart, FIG. 32F, adrenal glands; FIG. 32G, muscle; FIG. 32H, fat. Low dose of di-siRNA achieved the best silencing in lungs without silence the gene in other tissues. Intratracheal, 7.5 or 15 nmol, n=5, 1 week, bDNA QuantiGene assay.

FIG. 33 depicts target mRNA silencing (Htt) after intratracheal administration of mono and di-siRNAs. Intratracheal, 7.5 or 15 nmol, n=5, 1 week, bDNA QuantiGene assay.

FIG. 34 depicts a screen of siRNAs and ASOs targeting various SARS-CoV2 genes and tested for silencing efficacy. siRNAs and ASOs were tested in in A549-ACE2 cells and silencing was assessed using the psi-check reporter system. siRNA concentration: 10 nM; ASO concentration: 25 nM; Time point: 72 hours.

FIG. 35 depicts dose response data of select siRNAs targeting various SARS-CoV2 genes. The data reports relative mRNA abundance of the targeted SARS-CoV2 genes and the percent of cells that are positive for the SARS-CoV2 spike protein. siRNAs were tested in in A549-ACE2 cells and silencing was assessed using the psi-check reporter system.

FIG. 36 depicts dose response data of select siRNAs targeting various SARS-CoV2 genes. The A549-ACE2 cells were infected with SARS-CoV-2 at a MOI of 0.1 and 0.4. The data reports relative mRNA abundance of the targeted SARS-CoV2 genes.

FIG. 37 depicts a screen of siRNAs targeting the orf7a SARS-CoV2 gene. siRNAs were tested in in A549-ACE2 cells and the data reports relative mRNA abundance of the targeted orf7a SARS-CoV2 gene and the percent of cells that are positive for the SARS-CoV2 spike protein. siRNA concentration: 10 nM; Time point: 72 hours.

DETAILED DESCRIPTION

Novel SARS-CoV-2 target sequences are provided. Also provided are novel RNA molecules, such as siRNAs and branched RNA compounds containing the same, that target one or more SARS-CoV-2 genes mRNA, such as one or more target sequences of the disclosure. Also provided are novel ACE2, FURIN, IL-6, and TMPRSS2 target sequences.

Unless otherwise specified, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Unless otherwise specified, the methods and techniques provided herein are performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, delivery, and treatment of patients.

Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

So that the invention may be more readily understood, certain terms are first defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidine base covalently linked to a ribose or deoxyribose sugar. Exemplary nucleosides include adenosine, guanosine, cytidine, uridine and thymidine. Additional exemplary nucleosides include inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and N2,N2-dimethylguanosine (also referred to as “rare” nucleosides). The term “nucleotide” refers to a nucleoside having one or more phosphate groups joined in ester linkages to the sugar moiety. Exemplary nucleotides include nucleoside monophosphates, diphosphates and triphosphates. The terms “polynucleotide” and “nucleic acid molecule” are used interchangeably herein and refer to a polymer of nucleotides joined together by a phosphodiester or phosphorothioate linkage between 5′ and 3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refers to a polymer of ribonucleotides (e.g., 2, 3, 4, 5, 10, 15, 20, 25, 30, or more ribonucleotides). The term “DNA” or “DNA molecule” or “deoxyribonucleic acid molecule” refers to a polymer of deoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., by DNA replication or transcription of DNA, respectively). RNA can be post-transcriptionally modified. DNA and RNA can also be chemically synthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA, respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA and dsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNA that specifies the amino acid sequence of one or more polypeptide chains. This information is translated during protein synthesis when ribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (also referred to in the art as “short interfering RNAs”) refers to an RNA (or RNA analog) comprising between about 10-50 nucleotides (or nucleotide analogs), which is capable of directing or mediating RNA interference. In certain embodiments, a siRNA comprises between about 15-30 nucleotides or nucleotide analogs, or between about 16-25 nucleotides (or nucleotide analogs), or between about 18-23 nucleotides (or nucleotide analogs), or between about 19-22 nucleotides (or nucleotide analogs) (e.g., 19, 20, 21 or 22 nucleotides or nucleotide analogs). The term “short” siRNA refers to a siRNA comprising about 21 nucleotides (or nucleotide analogs), for example, 19, 20, 21 or 22 nucleotides. The term “long” siRNA refers to a siRNA comprising about 24-25 nucleotides, for example, 23, 24, 25 or 26 nucleotides. Short siRNAs may, in some instances, include fewer than 19 nucleotides, e.g., 16, 17 or 18 nucleotides, provided that the shorter siRNA retains the ability to mediate RNAi. Likewise, long siRNAs may, in some instances, include more than 26 nucleotides, provided that the longer siRNA retains the ability to mediate RNAi absent further processing, e.g., enzymatic processing, to a short siRNA.

The term “nucleotide analog” or “altered nucleotide” or “modified nucleotide” refers to a non-standard nucleotide, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position so as to alter certain chemical properties of the nucleotide yet retain the ability of the nucleotide analog to perform its intended function. Examples of positions of the nucleotide, which may be derivatized include: the 5 position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine, 5-propyne uridine, 5-propenyl uridine, etc.; the 6 position, e.g., 6-(2-amino)propyl uridine; and the 8-position for adenosine and/or guanosines, e.g., 8-bromo guanosine, 8-chloro guanosine, 8-fluoroguanosine, etc. Nucleotide analogs also include deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-modified (e.g., alkylated, e.g., N6-methyl adenosine, or as otherwise known in the art) nucleotides; and other heterocyclically modified nucleotide analogs, such as those described in Herdewijn, Antisense Nucleic Acid Drug Dev., 2000 Aug. 10(4):297-310.

Nucleotide analogs may also comprise modifications to the sugar portion of the nucleotides. For example, the 2′ OH-group may be replaced by a group selected from H, OR, R, F, Cl, Br, I, SH, SR, NH₂, NHR, NR₂, or COOR, wherein R is substituted or unsubstituted C₁-C₆ alkyl, alkenyl, alkynyl, aryl, etc. Other possible modifications include those described in U.S. Pat. Nos. 5,858,988, and 6,291,438.

The phosphate group of the nucleotide may also be modified, e.g., by substituting one or more of the oxygens of the phosphate group with sulfur (e.g., phosphorothioates), or by making other substitutions, which allow the nucleotide to perform its intended function, such as described in, for example, Eckstein, Antisense Nucleic Acid Drug Dev. 2000 Apr. 10(2):117-21, Rusckowski et al. Antisense Nucleic Acid Drug Dev. 2000 Oct. 10(5):333-45, Stein, Antisense Nucleic Acid Drug Dev. 2001 Oct. 11(5): 317-25, Vorobjev et al. Antisense Nucleic Acid Drug Dev. 2001 Apr. 11(2):77-85, and U.S. Pat. No. 5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) decrease the rate of hydrolysis of, for example, polynucleotides comprising said analogs in vivo or in vitro.

The term “oligonucleotide” refers to a short polymer of nucleotides and/or nucleotide analogs.

The term “RNA analog” refers to a polynucleotide (e.g., a chemically synthesized polynucleotide) having at least one altered or modified nucleotide as compared to a corresponding unaltered or unmodified RNA, but retaining the same or similar nature or function as the corresponding unaltered or unmodified RNA. As discussed above, the oligonucleotides may be linked with linkages, which result in a lower rate of hydrolysis of the RNA analog as compared to an RNA molecule with phosphodiester linkages. For example, the nucleotides of the analog may comprise methylenediol, ethylene diol, oxymethylthio, oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phosphoroamidate, and/or phosphorothioate linkages. Some RNA analogues include sugar- and/or backbone-modified ribonucleotides and/or deoxyribonucleotides. Such alterations or modifications can further include addition of non-nucleotide material, such as to the end(s) of the RNA or internally (at one or more nucleotides of the RNA). An RNA analog need only be sufficiently similar to natural RNA that it has the ability to mediate RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to a selective intracellular degradation of RNA. RNAi occurs in cells naturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAi proceeds via fragments cleaved from free dsRNA, which direct the degradative mechanism to other similar RNA sequences. Alternatively, RNAi can be initiated by the hand of man, for example, to silence the expression of target genes.

An RNAi agent, e.g., an RNA silencing agent, having a strand, which is “sequence sufficiently complementary to a target mRNA sequence to direct target-specific RNA interference (RNAi)” means that the strand has a sequence sufficient to trigger the destruction of the target mRNA by the RNAi machinery or process.

As used herein, the term “isolated RNA” (e.g., “isolated siRNA” or “isolated siRNA precursor”) refers to RNA molecules, which are substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term “RNA silencing” refers to a group of sequence-specific regulatory mechanisms (e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression) mediated by RNA molecules, which result in the inhibition or “silencing” of the expression of a corresponding protein-coding gene. RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.

The term “discriminatory RNA silencing” refers to the ability of an RNA molecule to substantially inhibit the expression of a “first” or “target” polynucleotide sequence while not substantially inhibiting the expression of a “second” or “non-target” polynucleotide sequence,” e.g., when both polynucleotide sequences are present in the same cell. In certain embodiments, the target polynucleotide sequence corresponds to a target gene, while the non-target polynucleotide sequence corresponds to a non-target gene. In other embodiments, the target polynucleotide sequence corresponds to a target allele, while the non-target polynucleotide sequence corresponds to a non-target allele. In certain embodiments, the target polynucleotide sequence is the DNA sequence encoding the regulatory region (e.g. promoter or enhancer elements) of a target gene. In other embodiments, the target polynucleotide sequence is a target mRNA encoded by a target gene.

The term “in vitro” has its art recognized meaning, e.g., involving purified reagents or extracts, e.g., cell extracts. The term “in vivo” also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and/or cells in an organism.

As used herein, the term “transgene” refers to any nucleic acid molecule, which is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from the cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism. The term “transgene” also means a nucleic acid molecule that includes one or more selected nucleic acid sequences, e.g., DNAs, that encode one or more engineered RNA precursors, to be expressed in a transgenic organism, e.g., animal, which is partly or entirely heterologous, i.e., foreign, to the transgenic animal, or homologous to an endogenous gene of the transgenic animal, but which is designed to be inserted into the animal's genome at a location which differs from that of the natural gene. A transgene includes one or more promoters and any other DNA, such as introns, necessary for expression of the selected nucleic acid sequence, all operably linked to the selected sequence, and may include an enhancer sequence.

A gene “involved” in a disease or disorder includes a gene, the normal or aberrant expression or function of which effects or causes the disease or disorder or at least one symptom of said disease or disorder.

The term “gain-of-function mutation” as used herein, refers to any mutation in a gene in which the protein encoded by said gene (i.e., the mutant protein) acquires a function not normally associated with the protein (i.e., the wild type protein) and causes or contributes to a disease or disorder. The gain-of-function mutation can be a deletion, addition, or substitution of a nucleotide or nucleotides in the gene, which gives rise to the change in the function of the encoded protein. In one embodiment, the gain-of-function mutation changes the function of the mutant protein or causes interactions with other proteins. In another embodiment, the gain-of-function mutation causes a decrease in or removal of normal wild-type protein, for example, by interaction of the altered, mutant protein with said normal, wild-type protein.

As used herein, the term “target gene” is a gene whose expression is to be substantially inhibited or “silenced.” This silencing can be achieved by RNA silencing, e.g., by cleaving the mRNA of the target gene or translational repression of the target gene. The term “non-target gene” is a gene whose expression is not to be substantially silenced. In one embodiment, the polynucleotide sequences of the target and non-target gene (e.g. mRNA encoded by the target and non-target genes) can differ by one or more nucleotides. In another embodiment, the target and non-target genes can differ by one or more polymorphisms (e.g., Single Nucleotide Polymorphisms or SNPs). In another embodiment, the target and non-target genes can share less than 100% sequence identity. In another embodiment, the non-target gene may be a homologue (e.g. an orthologue or paralogue) of the target gene.

As described herein, the term “SARS-CoV-2” refers to the severe acute respiratory syndrome coronavirus 2, which can cause severe respiratory illness. The SARS-CoV-2 genome contains nine genes. Four genes encode for four structural proteins, S (spike), E (envelope), M (matrix) and N (nucleocapsid). The five other genes are orfla, orflab, 3a, 8b, and 7a. The sequence of the SARS-CoV-2 and the nine genes are recited in Table 1 and Table 2, respectively.

The phrase “examining the function of a gene in a cell or organism” refers to examining or studying the expression, activity, function or phenotype arising therefrom.

As used herein, the term “RNA silencing agent” refers to an RNA, which is capable of inhibiting or “silencing” the expression of a target gene. In certain embodiments, the RNA silencing agent is capable of preventing complete processing (e.g., the full translation and/or expression) of a mRNA molecule through a post-transcriptional silencing mechanism. RNA silencing agents include small (<50 b.p.), noncoding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small noncoding RNAs can be generated. Exemplary RNA silencing agents include siRNAs, miRNAs, siRNA-like duplexes, antisense oligonucleotides, GAPMER molecules, and dual-function oligonucleotides, as well as precursors thereof. In one embodiment, the RNA silencing agent is capable of inducing RNA interference. In another embodiment, the RNA silencing agent is capable of mediating translational repression.

As used herein, the term “rare nucleotide” refers to a naturally occurring nucleotide that occurs infrequently, including naturally occurring deoxyribonucleotides or ribonucleotides that occur infrequently, e.g., a naturally occurring ribonucleotide that is not guanosine, adenosine, cytosine, or uridine. Examples of rare nucleotides include, but are not limited to, inosine, 1-methyl inosine, pseudouridine, 5,6-dihydrouridine, ribothymidine, 2N-methylguanosine and 2,2N,N-dimethylguanosine.

The term “engineered,” as in an engineered RNA precursor, or an engineered nucleic acid molecule, indicates that the precursor or molecule is not found in nature, in that all or a portion of the nucleic acid sequence of the precursor or molecule is created or selected by a human. Once created or selected, the sequence can be replicated, translated, transcribed, or otherwise processed by mechanisms within a cell. Thus, an RNA precursor produced within a cell from a transgene that includes an engineered nucleic acid molecule is an engineered RNA precursor.

As used herein, the term “microRNA” (“miRNA”), also known in the art as “small temporal RNAs” (“stRNAs”), refers to a small (10-50 nucleotide) RNA, which are genetically encoded (e.g., by viral, mammalian, or plant genomes) and are capable of directing or mediating RNA silencing. An “miRNA disorder” shall refer to a disease or disorder characterized by an aberrant expression or activity of a miRNA.

As used herein, the term “dual functional oligonucleotide” refers to a RNA silencing agent having the formula T-L-μ, wherein T is an mRNA targeting moiety, L is a linking moiety, and p is a miRNA recruiting moiety. As used herein, the terms “mRNA targeting moiety,” “targeting moiety,” “mRNA targeting portion” or “targeting portion” refer to a domain, portion or region of the dual functional oligonucleotide having sufficient size and sufficient complementarity to a portion or region of an mRNA chosen or targeted for silencing (i.e., the moiety has a sequence sufficient to capture the target mRNA).

As used herein, the term “linking moiety” or “linking portion” refers to a domain, portion or region of the RNA-silencing agent which covalently joins or links the mRNA.

As used herein, the term “antisense strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is substantially complementary to a section of about 10-50 nucleotides, e.g., about 15-30, 16-25, 18-23 or 19-22 nucleotides of the mRNA of the gene targeted for silencing. The antisense strand or first strand has sequence sufficiently complementary to the desired target mRNA sequence to direct target-specific silencing, e.g., complementarity sufficient to trigger the destruction of the desired target mRNA by the RNAi machinery or process (RNAi interference) or complementarity sufficient to trigger translational repression of the desired target mRNA.

The term “sense strand” or “second strand” of an RNA silencing agent, e.g., an siRNA or RNA silencing agent, refers to a strand that is complementary to the antisense strand or first strand. Antisense and sense strands can also be referred to as first or second strands, the first or second strand having complementarity to the target sequence and the respective second or first strand having complementarity to said first or second strand. miRNA duplex intermediates or siRNA-like duplexes include a miRNA strand having sufficient complementarity to a section of about 10-50 nucleotides of the mRNA of the gene targeted for silencing and a miRNA* strand having sufficient complementarity to form a duplex with the miRNA strand.

As used herein, the term “guide strand” refers to a strand of an RNA silencing agent, e.g., an antisense strand of an siRNA duplex or siRNA sequence, that enters into the RISC complex and directs cleavage of the target mRNA.

As used herein, the term “asymmetry,” as in the asymmetry of the duplex region of an RNA silencing agent (e.g., the stem of an shRNA), refers to an inequality of bond strength or base pairing strength between the termini of the RNA silencing agent (e.g., between terminal nucleotides on a first strand or stem portion and terminal nucleotides on an opposing second strand or stem portion), such that the 5′ end of one strand of the duplex is more frequently in a transient unpaired, e.g., single-stranded, state than the 5′ end of the complementary strand. This structural difference determines that one strand of the duplex is preferentially incorporated into a RISC complex. The strand whose 5′ end is less tightly paired to the complementary strand will preferentially be incorporated into RISC and mediate RNAi.

As used herein, the term “bond strength” or “base pair strength” refers to the strength of the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., an siRNA duplex), due primarily to H-bonding, van der Waals interactions, and the like, between said nucleotides (or nucleotide analogs).

As used herein, the “5′ end,” as in the 5′ end of an antisense strand, refers to the 5′ terminal nucleotides, e.g., between one and about 5 nucleotides at the 5′ terminus of the antisense strand. As used herein, the “3′ end,” as in the 3′ end of a sense strand, refers to the region, e.g., a region of between one and about 5 nucleotides, that is complementary to the nucleotides of the 5′ end of the complementary antisense strand.

As used herein the term “destabilizing nucleotide” refers to a first nucleotide or nucleotide analog capable of forming a base pair with second nucleotide or nucleotide analog such that the base pair is of lower bond strength than a conventional base pair (i.e., Watson-Crick base pair). In certain embodiments, the destabilizing nucleotide is capable of forming a mismatch base pair with the second nucleotide. In other embodiments, the destabilizing nucleotide is capable of forming a wobble base pair with the second nucleotide. In yet other embodiments, the destabilizing nucleotide is capable of forming an ambiguous base pair with the second nucleotide.

As used herein, the term “base pair” refers to the interaction between pairs of nucleotides (or nucleotide analogs) on opposing strands of an oligonucleotide duplex (e.g., a duplex formed by a strand of a RNA silencing agent and a target mRNA sequence), due primarily to H-bonding, van der Waals interactions, and the like between said nucleotides (or nucleotide analogs). As used herein, the term “bond strength” or “base pair strength” refers to the strength of the base pair.

As used herein, the term “mismatched base pair” refers to a base pair consisting of non-complementary or non-Watson-Crick base pairs, for example, not normal complementary G:C, A:T or A:U base pairs. As used herein the term “ambiguous base pair” (also known as a non-discriminatory base pair) refers to a base pair formed by a universal nucleotide.

As used herein, term “universal nucleotide” (also known as a “neutral nucleotide”) include those nucleotides (e.g. certain destabilizing nucleotides) having a base (a “universal base” or “neutral base”) that does not significantly discriminate between bases on a complementary polynucleotide when forming a base pair. Universal nucleotides are predominantly hydrophobic molecules that can pack efficiently into antiparallel duplex nucleic acids (e.g., double-stranded DNA or RNA) due to stacking interactions. The base portion of universal nucleotides typically comprise a nitrogen-containing aromatic heterocyclic moiety.

As used herein, the terms “sufficient complementarity” or “sufficient degree of complementarity” mean that the RNA silencing agent has a sequence (e.g. in the antisense strand, mRNA targeting moiety or miRNA recruiting moiety), which is sufficient to bind the desired target RNA, respectively, and to trigger the RNA silencing of the target mRNA.

As used herein, the term “translational repression” refers to a selective inhibition of mRNA translation. Natural translational repression proceeds via miRNAs cleaved from shRNA precursors. Both RNAi and translational repression are mediated by RISC. Both RNAi and translational repression occur naturally or can be initiated by the hand of man, for example, to silence the expression of target genes.

Various methodologies of the instant invention include a step that involves comparing a value, level, feature, characteristic, property, etc. to a “suitable control,” referred to interchangeably herein as an “appropriate control.” A “suitable control” or “appropriate control” is any control or standard familiar to one of ordinary skill in the art useful for comparison purposes. In one embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined prior to performing an RNAi methodology, as described herein. For example, a transcription rate, mRNA level, translation rate, protein level, biological activity, cellular characteristic or property, genotype, phenotype, etc. can be determined prior to introducing an RNA silencing agent of the invention into a cell or organism. In another embodiment, a “suitable control” or “appropriate control” is a value, level, feature, characteristic, property, etc. determined in a cell or organism, e.g., a control or normal cell or organism, exhibiting, for example, normal traits. In yet another embodiment, a “suitable control” or “appropriate control” is a predefined value, level, feature, characteristic, property, etc.

As used herein, the terms “extended nucleic acid” or “exNA” or ex-NA” refer to a novel oligonucleotide backbone modification. This chemical modification of the backbone significantly enhances oligonucleotide metabolic stability. The chemical modification includes one or more carbon atoms or chains inserted in the backbone at the 5′-position, 3′-position, or both. This structural modulation forms non-canonical stretched/flexible structure on oligo-backbones, which protect oligonucleotides from cleavage by various nucleases.

The novel exNA-modification is widely compatible in any oligonucleotide, such as an siRNA, antisense oligonucleotide, and mRNA. The combination of an exNA-phosphorothioate (exNA-PS) backbone enables drastic enhancement of metabolic stability (10-50 orders of magnitude as compared to unmodified oligonucleotides) without compromising the function of the oligonucleotide (e.g., siRNA-mediated silencing efficacy). For example, 5′-[exNA-PS]4-3′ modification induce NO negative impact on siRNA efficacy while inducing drastically high exonuclease stability, as will be shown below. Moreover, an exNA-phosphodiester (exNA-PO) backbone also enables drastic enhancement of metabolic stability without compromising the function of the oligonucleotide. It has been previously shown that phosphorothioate-containing backbones in oligonucleotides are toxic when administered in vivo. Accordingly, the exNA-PO backbone can be employed to enhancement of metabolic stability while decreasing toxicity. Thus, this metabolically stabilizing exNA modification is widely and robustly improves the performance of therapeutic oligonucleotide candidates in vivo.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and example are illustrative only and not intended to be limiting.

Various aspects of the invention are described in further detail in the following subsections.

I. Novel Target Sequences

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting a SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10, as recited in Table 4 and Table 5.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 6A. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 6A.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 7. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 7.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 8. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 8.

In certain exemplary embodiments, RNA silencing agents of the invention comprise an antisense strand as recited in Table 6B. In certain exemplary embodiments, RNA silencing agents of the invention comprise a sense strand as recited in Table 6C. In certain exemplary embodiments, RNA silencing agents of the invention comprise a sense strand as recited in Table 6D.

In one embodiment, the RNA silencing agents of the invention capable of targeting one or more of a SARS-CoV-2 nucleic acid sequence can be combined with RNA silencing agents of the invention capable of targeting one or more of an ACE2, FURIN, TMPRSS2, IL-6, and IL-6R nucleic acid sequence. The endogenous genes ACE2, FURIN, TMPRSS2, IL-6, and IL-6R each may play a role in SARS-CoV-2 infection and pathogenesis.

“ACE2”, as described herein, refers to angiotensin I converting enzyme 2. ACE2 belongs to a family of dipeptidyl carboxydipeptidases and is a zinc-containing metallo protease. ACE2 cleaves angiotensin I into angiotensin II, which has vasoconstrictive properties. It also is a functional receptor of the spike glycoprotein of the human corona viruses. ACE2 expression is age and disease state dependent. Children have lower ACE2 expression, which may explain their decreased susceptibility and milder disease symptoms upon SARS-CoV-2 infection. As such, a reduction in ACE2 expression is a viable therapeutic approach.

“FURIN”, as described herein, refers to a subtilisin-like proprotein belonging to the convertase family of proteases. They include proteases that process protein and peptides precursors as they traffic through the constitute branches of the secretory pathway. Furin is exploited by viruses for cleaving envelope proteins. The spike protein of the SARS-CoV-2 virus must be cleaved by furin to become functional, and as such furin represents an attractive target for siRNA (Coutard et al. Antiviral Research. 176: 104727. April 2020).

“TMPRSS2”, as described herein, refers to Transmembrane Serine Protease 2. TMPRSS2 has been shown to contribute to virus spread and immunopathology in the airways of murine models after coronavirus infection (Iwata-Yoshikawa et al. J. Virol. 93 (6): e01815-18. March 2019).

“IL-6”, as described herein, refers to interleukin-6. “IL-6R”, as described herein, refers to interleukin-6 receptor. IL-6 is an inflammatory agent that contributes to cytokine release syndrome (CRS), a severe and potentially deadly response to an infection. IL-6 stimulates inflammation through an interaction with IL-6R (Liu et al. J Autoimmun. 10: 102452. April 2020). Inhibition of IL-6 or its receptor, IL-6R may prevent or reduce the cytokine release syndrome that occurs during a SARS-CoV-2 infection.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an ACE2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 11D and Table 12C. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an ACE2 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 1 ID and Table 12C.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an FURIN nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 11C and Table 12D. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an FURIN nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 1 IC and Table 12D.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an TMPRSS2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 11A and Table 12A. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an TMPRSS2 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 11A and Table 12A.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an IL-6 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 11B and Table 12B. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an IL-6 nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 11B and Table 12B.

In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an IL-6R nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 12E. In certain exemplary embodiments, RNA silencing agents of the invention are capable of targeting an IL-6R nucleic acid sequence of any one of the 20-nucleotide target regions recited in Table 12E.

Genomic sequence for each target sequence can be found in, for example, the publicly available database maintained by the NCBI.

II. siRNA Design

In some embodiments, siRNAs are designed as follows. First, a portion of the target gene (e.g., the SARS-CoV-2 gene), e.g., one or more of the target sequences set forth in Table 4, Table 5, Table 6A, Table 7, or Table 8 is selected. Cleavage of mRNA at these sites should eliminate translation of corresponding protein. Antisense strands were designed based on the target sequence and sense strands were designed to be complementary to the antisense strand. Hybridization of the antisense and sense strands forms the siRNA duplex. The antisense strand includes about 19 to 25 nucleotides, e.g., 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the antisense strand includes 20, 21, 22 or 23 nucleotides. The sense strand includes about 14 to 25 nucleotides, e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In other embodiments, the sense strand is 15 nucleotides. In other embodiments, the sense strand is 18 nucleotides. In other embodiments, the sense strand is 20 nucleotides. The skilled artisan will appreciate, however, that siRNAs having a length of less than 19 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant invention, provided that they retain the ability to mediate RNAi. Longer RNAi agents have been demonstrated to elicit an interferon or PKR response in certain mammalian cells, which may be undesirable. In certain embodiments, the RNAi agents of the invention do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNAi agents may be useful, for example, in cell types incapable of generating a PKR response or in situations where the PKR response has been down-regulated or dampened by alternative means.

The sense strand sequence can be designed such that the target sequence is essentially in the middle of the strand. Moving the target sequence to an off-center position can, in some instances, reduce efficiency of cleavage by the siRNA. Such compositions, i.e., less efficient compositions, may be desirable for use if off-silencing of the wild-type mRNA is detected.

The antisense strand can be the same length as the sense strand and includes complementary nucleotides. In one embodiment, the strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands align or anneal such that 1-, 2-, 3-, 4-, 5-, 6-, 7-, or 8-nucleotide overhangs are generated, i.e., the 3′ end of the sense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides further than the 5′ end of the antisense strand and/or the 3′ end of the antisense strand extends 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides further than the 5′ end of the sense strand. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material.

To facilitate entry of the antisense strand into RISC (and thus increase or improve the efficiency of target cleavage and silencing), the base pair strength between the 5′ end of the sense strand and 3′ end of the antisense strand can be altered, e.g., lessened or reduced, as described in detail in U.S. Pat. Nos. 7,459,547, 7,772,203 and 7,732,593, entitled “Methods and Compositions for Controlling Efficacy of RNA Silencing” (filed Jun. 2, 2003) and U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705, entitled “Methods and Compositions for Enhancing the Efficacy and Specificity of RNAi” (filed Jun. 2, 2003), the contents of which are incorporated in their entirety by this reference. In one embodiment of these aspects of the invention, the base-pair strength is less due to fewer G:C base pairs between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand than between the 3′ end of the first or antisense strand and the 5′ end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. In certain exemplary embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the base pair strength is less due to at least one wobble base pair, e.g., G:U, between the 5′ end of the first or antisense strand and the 3′ end of the second or sense strand. In another embodiment, the base pair strength is less due to at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain exemplary embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the base pair strength is less due to at least one base pair comprising a modified nucleotide. In certain exemplary embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

The design of siRNAs suitable for targeting the SARS-Co V-2 target sequences set forth in Table 4, Table 5, Table 6A, Table 7, or Table 8 is described in detail below. siRNAs can be designed according to the above exemplary teachings for any other target sequences found in the SARS-CoV-2 gene. Moreover, the technology is applicable to targeting any other target sequences, e.g., non-disease-causing target sequences.

To validate the effectiveness by which siRNAs destroy mRNAs (e.g., SARS-CoV-2 mRNA), the siRNA can be incubated with cDNA (e.g., SARS-CoV-2 cDNA) in a Drosophila-based in vitro mRNA expression system. Radiolabeled with ³²P, newly synthesized mRNAs (e.g., SARS-CoV-2 mRNA) are detected autoradiographically on an agarose gel. The presence of cleaved mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA; a homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence. Sites of siRNA-mRNA complementation are selected which result in optimal mRNA specificity and maximal mRNA cleavage.

III. RNAi Agents

The present invention includes RNAi molecules, such as siRNA molecules designed, for example, as described above. The siRNA molecules of the invention can be chemically synthesized, or can be transcribed in vitro from a DNA template, or in vivo from e.g., shRNA, or by using recombinant human DICER enzyme, to cleave in vitro transcribed dsRNA templates into pools of 20-, 21- or 23-bp duplex RNA mediating RNAi. The siRNA molecules can be designed using any method known in the art.

In one aspect, instead of the RNAi agent being an interfering ribonucleic acid, e.g., an siRNA or shRNA as described above, the RNAi agent can encode an interfering ribonucleic acid, e.g., an shRNA, as described above. In other words, the RNAi agent can be a transcriptional template of the interfering ribonucleic acid. Thus, RNAi agents of the present invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3′ UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21-23 nucleotides (Brummelkamp et al., 2002; Lee et al., 2002, Supra; Miyagishi et al., 2002; Paddison et al., 2002, supra; Paul et al., 2002, supra; Sui et al., 2002 supra; Yu et al., 2002, supra. More information about shRNA design and use can be found on the internet at the following addresses: katandin.cshl.org:9331/RNAi/docs/BseRI-BamHI_Strategy.pdf and katandin.cshl.org:9331/RNAi/docs/Web_version_of_PCR_strategyl.pdf).

Expression constructs of the present invention include any construct suitable for use in the appropriate expression system and include, but are not limited to, retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs can include one or more inducible promoters, RNA Pol III promoter systems, such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct. (Tuschl, T., 2002, Supra).

Synthetic siRNAs can be delivered into cells by methods known in the art, including cationic liposome transfection and electroporation. To obtain longer term suppression of the target genes (e.g., SARS-CoV-2 genes) and to facilitate delivery under certain circumstances, one or more siRNA can be expressed within cells from recombinant DNA constructs. Such methods for expressing siRNA duplexes within cells from recombinant DNA constructs to allow longer-term target gene suppression in cells are known in the art, including mammalian Pol III promoter systems (e.g., H1 or U6/snRNA promoter systems (Tuschl, T., 2002, supra) capable of expressing functional double-stranded siRNAs; (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002, supra; Sui et al., 2002, supra). Transcriptional termination by RNA Pol III occurs at runs of four consecutive T residues in the DNA template, providing a mechanism to end the siRNA transcript at a specific sequence. The siRNA is complementary to the sequence of the target gene in 5′-3′ and 3′-5′ orientations, and the two strands of the siRNA can be expressed in the same construct or in separate constructs. Hairpin siRNAs, driven by H1 or U6 snRNA promoter and expressed in cells, can inhibit target gene expression (Bagella et al., 1998; Lee et al., 2002, supra; Miyagishi et al., 2002, supra; Paul et al., 2002, supra; Yu et al., 2002), supra; Sui et al., 2002, supra). Constructs containing siRNA sequence under the control of T7 promoter also make functional siRNAs when co-transfected into the cells with a vector expressing T7 RNA polymerase (Jacque et al., 2002, supra). A single construct may contain multiple sequences coding for siRNAs, such as multiple regions of the gene encoding SARS-CoV-2, targeting the same gene or multiple genes, and can be driven, for example, by separate PolIII promoter sites.

Animal cells express a range of noncoding RNAs of approximately 22 nucleotides termed micro RNA (miRNAs), which can regulate gene expression at the post transcriptional or translational level during animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. By substituting the stem sequences of the miRNA precursor with sequence complementary to the target mRNA, a vector construct that expresses the engineered precursor can be used to produce siRNAs to initiate RNAi against specific mRNA targets in mammalian cells (Zeng et al., 2002, supra). When expressed by DNA vectors containing polymerase III promoters, micro-RNA designed hairpins can silence gene expression (McManus et al., 2002, supra). MicroRNAs targeting polymorphisms may also be useful for blocking translation of mutant proteins, in the absence of siRNA-mediated gene-silencing. Such applications may be useful in situations, for example, where a designed siRNA caused off-target silencing of wild type protein.

Viral-mediated delivery mechanisms can also be used to induce specific silencing of targeted genes through expression of siRNA, for example, by generating recombinant adenoviruses harboring siRNA under RNA Pol II promoter transcription control (Xia et al., 2002, supra). Infection of HeLa cells by these recombinant adenoviruses allows for diminished endogenous target gene expression. Injection of the recombinant adenovirus vectors into transgenic mice expressing the target genes of the siRNA results in in vivo reduction of target gene expression. Id. In an animal model, whole-embryo electroporation can efficiently deliver synthetic siRNA into post-implantation mouse embryos (Calegari et al., 2002). In adult mice, efficient delivery of siRNA can be accomplished by “high-pressure” delivery technique, a rapid injection (within 5 seconds) of a large volume of siRNA containing solution into animal via the tail vein (Liu et al., 1999, supra; McCaffrey et al., 2002, supra; Lewis et al., 2002. Nanoparticles and liposomes can also be used to deliver siRNA into animals. In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., lung (e.g., endothelial cells, epithelial cells, fibroblasts, and immune cells in the lungs, e.g. clara cells, alveolar cells, and club cells)

The nucleic acid compositions of the invention include both unmodified siRNAs and modified siRNAs, such as crosslinked siRNA derivatives or derivatives having non-nucleotide moieties linked, for example to their 3′ or 5′ ends. Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative, as compared to the corresponding siRNA, and are useful for tracing the siRNA derivative in the cell, or improving the stability of the siRNA derivative compared to the corresponding siRNA.

Engineered RNA precursors, introduced into cells or whole organisms as described herein, will lead to the production of a desired siRNA molecule. Such an siRNA molecule will then associate with endogenous protein components of the RNAi pathway to bind to and target a specific mRNA sequence for cleavage and destruction. In this fashion, the mRNA, which will be targeted by the siRNA generated from the engineered RNA precursor, and will be depleted from the cell or organism, leading to a decrease in the concentration of the protein encoded by that mRNA in the cell or organism. The RNA precursors are typically nucleic acid molecules that individually encode either one strand of a dsRNA or encode the entire nucleotide sequence of an RNA hairpin loop structure.

The nucleic acid compositions of the invention can be unconjugated or can be conjugated to another moiety, such as a nanoparticle, to enhance a property of the compositions, e.g., a pharmacokinetic parameter such as absorption, efficacy, bioavailability and/or half-life. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to poly alkylcy anoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53 (1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeled using any method known in the art. For instance, the nucleic acid compositions can be labeled with a fluorophore, e.g., Cy3, fluorescein, or rhodamine. The labeling can be carried out using a kit, e.g., the SILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can be radiolabeled, e.g., using ³H, ³²P or another appropriate isotope.

Moreover, because RNAi is believed to progress via at least one single-stranded RNA intermediate, the skilled artisan will appreciate that ss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also be designed (e.g., for chemical synthesis), generated (e.g., enzymatically generated), or expressed (e.g., from a vector or plasmid) as described herein and utilized according to the claimed methodologies. Moreover, in invertebrates, RNAi can be triggered effectively by long dsRNAs (e.g., dsRNAs about 100-1000 nucleotides in length, such as about 200-500, for example, about 250, 300, 350, 400 or 450 nucleotides in length) acting as effectors of RNAi. (Brondani et al., Proc Natl Acad Sci USA. 2001 Dec. 4; 98(25):14428-33. Epub 2001 Nov. 27.)

IV. Anti-SARS-CoV-2 RNA Silencing Agents

In certain embodiment, the present invention provides novel anti-SARS-CoV-2 RNA silencing agents (e.g., siRNA, shRNA, and antisense oligonucleotides), methods of making said RNA silencing agents, and methods (e.g., research and/or therapeutic methods) for using said improved RNA silencing agents (or portions thereof) for RNA silencing of SARS-CoV-2 protein. The RNA silencing agents comprise an antisense strand (or portions thereof), wherein the antisense strand has sufficient complementary to a target SARS-CoV-2 mRNA to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

In certain embodiments, siRNA compounds are provided having one or any combination of the following properties: (1) fully chemically-stabilized (i.e., no unmodified 2′-OH residues); (2) asymmetry; (3) 11-20 base pair duplexes; (4) greater than 50% 2′-methoxy modifications, such as 70%-100% 2′-methoxy modifications, although an alternating pattern of chemically-modified nucleotides (e.g., 2′-fluoro and 2′-methoxy modifications), are also contemplated; and (5) single-stranded, fully phosphorothioated tails of 5-8 bases. In certain embodiments, the number of phosphorothioate modifications is varied from 4 to 16 total. In certain embodiments, the number of phosphorothioate modifications is varied from 8 to 13 total.

In certain embodiments, the siRNA compounds described herein can be conjugated to a variety of targeting agents, including, but not limited to, docosanoic acid (DCA), cholesterol, docosahexaenoic acid (DHA), phenyltropanes, cortisol, vitamin A, vitamin D, N-acetylgalactosamine (GalNac), and gangliosides. The cholesterol-modified version showed 5-10 fold improvement in efficacy in vitro versus previously used chemical stabilization patterns (e.g., wherein all purine but not pyrimidines are modified) in wide range of cell types (e.g., HeLa, neurons, hepatocytes, trophoblasts. lung epithelial cells).

Certain compounds of the invention having the structural properties described above and herein may be referred to as “hsiRNA-ASP” (hydrophobically-modified, small interfering RNA, featuring an advanced stabilization pattern). In addition, siRNAs conjugated to DCA or EPA and containing different numbers of 3′ exNA modifications and phosphorothioates showed a dramatically improved distribution through the lung, making them accessible for therapeutic intervention.

In certain embodiments, the siRNA comprises between 6 and 13 total phosphorothioate modifications. In certain embodiments, the siRNA comprises 6 phosphorothioate modifications. In certain embodiments, the siRNA comprises 8 phosphorothioate modifications. In certain embodiments, the siRNA antisense strand comprises 6 phosphorothioate modifications. In certain embodiments, the siRNA antisense strand comprises 4 phosphorothioate modifications. In certain embodiments, the siRNA sense strand comprises 2 phosphorothioate modifications.

In certain embodiments, the siRNA sense strand 3′ end is conjugated to DCA. In certain embodiments, the siRNA sense strand 3′ end is conjugated to EPA.

In certain embodiments, the siRNA antisense strand comprises two to five 3′ exNA modifications. In certain embodiments, the siRNA antisense strand comprises two 3′ exNA modifications. In certain embodiments, the siRNA antisense strand comprises three 3′ exNA modifications. In certain embodiments, the siRNA antisense strand comprises four 3′ exNA modifications. In certain embodiments, the siRNA antisense strand comprises five 3′ exNA modifications. In certain embodiments, the siRNA antisense strand comprises four consecutive 3′ exNA modifications.

In certain embodiments, the siRNA antisense strand comprises two to five 3′ exNA modifications and 6 phosphorothioate modifications. In certain embodiments, the siRNA antisense strand comprises two to five 3′ exNA modifications and 4 phosphorothioate modifications. In certain embodiments, the siRNA antisense strand comprises four 3′ exNA modifications and 6 phosphorothioate modifications. In certain embodiments, the siRNA antisense strand comprises four 3′ exNA modifications and 4 phosphorothioate modifications.

The compounds of the invention can be described in the following aspects and embodiments.

In a first aspect, provided herein is a double stranded RNA (dsRNA) comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides;

(3) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; and

(7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a second aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 70% 2′-O-methyl modifications;

(3) the nucleotide at position 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises at least 70% 2′-O-methyl modifications; and

(7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a third aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 85% 2′-O-methyl modifications;

(3) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises 100% 2′-O-methyl modifications; and

(7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a fourth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(3) the nucleotides at positions 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises 100% 2′-O-methyl modifications; and

(7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a fifth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(3) the nucleotides at positions 2, 4, 5, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises 100% 2′-O-methyl modifications; and

(7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a sixth aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 7, 9, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and

(8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

In a seventh aspect, provided herein is a dsRNA comprising an antisense strand and a sense strand, each strand comprising at least 14 contiguous nucleotides, with a 5′ end and a 3′ end, wherein:

(1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A;

(2) the antisense strand comprises at least 75% 2′-O-methyl modifications;

(3) the nucleotides at positions 2, 6, and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides;

(4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages;

(5) a portion of the antisense strand is complementary to a portion of the sense strand;

(6) the sense strand comprises at least 80% 2′-O-methyl modifications;

(7) the nucleotides at positions 7, 10, and 11 from the 3′ end of the sense strand are not 2′-methoxy-ribonucleotides; and

(8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages.

a) Design of Anti-SARS-CoV-2 siRNA Molecules

An siRNA molecule of the application is a duplex made of a sense strand and complementary antisense strand, the antisense strand having sufficient complementary to a SARS-CoV-2 mRNA to mediate RNAi. In certain embodiments, the siRNA molecule has a length from about 10-50 or more nucleotides, i.e., each strand comprises 10-50 nucleotides (or nucleotide analogs). In other embodiments, the siRNA molecule has a length from about 15-30, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in each strand, wherein one of the strands is sufficiently complementary to a target region. In certain embodiments, the strands are aligned such that there are at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 bases at the end of the strands, which do not align (i.e., for which no complementary bases occur in the opposing strand), such that an overhang of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues occurs at one or both ends of the duplex when strands are annealed.

Usually, siRNAs can be designed by using any method known in the art, for instance, by using the following protocol:

1. The siRNA should be specific for a target sequence, e.g., a target sequence set forth in the Examples. The first strand should be complementary to the target sequence, and the other strand is substantially complementary to the first strand. (See Examples for exemplary sense and antisense strands.) Exemplary target sequences are selected from any region of the target gene that leads to potent gene silencing. Regions of the target gene include, but are not limited to, the 5′ untranslated region (5′-UTR) of a target gene, the 3′ untranslated region (3′-UTR) of a target gene, an exon of a target gene, or an intron of a target gene. Cleavage of mRNA at these sites should eliminate translation of corresponding SARS-CoV-2 protein. Target sequences from other regions of the SARS-CoV-2 gene are also suitable for targeting. A sense strand is designed based on the target sequence.

2. The sense strand of the siRNA is designed based on the sequence of the selected target site. In certain embodiments, the sense strand includes about 15 to 25 nucleotides, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides. In certain embodiments, the sense strand includes 15, 16, 17, 18, 19, or 20 nucleotides. In certain embodiments, the sense strand is 15 nucleotides in length. In certain embodiments, the sense strand is 18 nucleotides in length. In certain embodiments, the sense strand is 20 nucleotides in length. The skilled artisan will appreciate, however, that siRNAs having a length of less than 15 nucleotides or greater than 25 nucleotides can also function to mediate RNAi. Accordingly, siRNAs of such length are also within the scope of the instant invention, provided that they retain the ability to mediate RNAi. Longer RNA silencing agents have been demonstrated to elicit an interferon or Protein Kinase R (PKR) response in certain mammalian cells which may be undesirable. In certain embodiments, the RNA silencing agents of the invention do not elicit a PKR response (i.e., are of a sufficiently short length). However, longer RNA silencing agents may be useful, for example, in cell types incapable of generating a PKR response or in situations where the PKR response has been down-regulated or dampened by alternative means.

The siRNA molecules of the invention have sufficient complementarity with the target sequence such that the siRNA can mediate RNAi. In general, siRNA containing nucleotide sequences sufficiently complementary to a target sequence portion of the target gene to effect RISC-mediated cleavage of the target gene are contemplated. Accordingly, in a certain embodiment, the antisense strand of the siRNA is designed to have a sequence sufficiently complementary to a portion of the target. For example, the antisense strand may have 100% complementarity to the target site. However, 100% complementarity is not required. Greater than 80% identity, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or even 100% complementarity, between the antisense strand and the target RNA sequence is contemplated. The present application has the advantage of being able to tolerate certain sequence variations to enhance efficiency and specificity of RNAi. In one embodiment, the antisense strand has 4, 3, 2, 1, or 0 mismatched nucleotide(s) with a target region, such as a target region that differs by at least one base pair between a wild-type and mutant allele, e.g., a target region comprising the gain-of-function mutation, and the other strand is identical or substantially identical to the first strand. Moreover, siRNA sequences with small insertions or deletions of 1 or 2 nucleotides may also be effective for mediating RNAi. Alternatively, siRNA sequences with nucleotide analog substitutions or insertions can be effective for inhibition.

Sequence identity may be determined by sequence comparison and alignment algorithms known in the art. To determine the percent identity of two nucleic acid sequences (or of two amino acid sequences), the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the first sequence or second sequence for optimal alignment). The nucleotides (or amino acid residues) at corresponding nucleotide (or amino acid) positions are then compared. When a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=number of identical positions/total number of positions×100), optionally penalizing the score for the number of gaps introduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In one embodiment, the alignment generated over a certain portion of the sequence aligned having sufficient identity but not over portions having low degree of identity (i.e., a local alignment). A non-limiting example of a local alignment algorithm utilized for the comparison of sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithm is incorporated into the BLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducing appropriate gaps and the percent identity is determined over the length of the aligned sequences (i.e., a gapped alignment). To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. In another embodiment, the alignment is optimized by introducing appropriate gaps and percent identity is determined over the entire length of the sequences aligned (i.e., a global alignment). A non-limiting example of a mathematical algorithm utilized for the global comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989). Such an algorithm is incorporated into the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

3. The antisense or guide strand of the siRNA is routinely the same length as the sense strand and includes complementary nucleotides. In one embodiment, the guide and sense strands are fully complementary, i.e., the strands are blunt-ended when aligned or annealed. In another embodiment, the strands of the siRNA can be paired in such a way as to have a 3′ overhang of 1 to 7 (e.g., 2, 3, 4, 5, 6 or 7), or 1 to 4, e.g., 2, 3 or 4 nucleotides. Overhangs can comprise (or consist of) nucleotides corresponding to the target gene sequence (or complement thereof). Alternatively, overhangs can comprise (or consist of) deoxyribonucleotides, for example dTs, or nucleotide analogs, or other suitable non-nucleotide material. Thus, in another embodiment, the nucleic acid molecules may have a 3′ overhang of 2 nucleotides, such as TT. The overhanging nucleotides may be either RNA or DNA. As noted above, it is desirable to choose a target region wherein the mutant:wild type mismatch is a purine:purine mismatch.

4. Using any method known in the art, compare the potential targets to the appropriate genome database (human, mouse, rat, etc.) and eliminate from consideration any target sequences with significant homology to other coding sequences. One such method for such sequence homology searches is known as BLAST, which is available at National Center for Biotechnology Information website.

5. Select one or more sequences that meet your criteria for evaluation.

Further general information about the design and use of siRNA may be found in “The siRNA User Guide,” available at The Max-Plank-Institut fur Biophysikalische Chemie website.

Alternatively, the siRNA may be defined functionally as a nucleotide sequence (or oligonucleotide sequence) that is capable of hybridizing with the target sequence (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. hybridization for 12-16 hours; followed by washing). Additional hybridization conditions include hybridization at 70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at 70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in 4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (T_(m)) of the hybrid, where T_(m) is determined according to the following equations. For hybrids less than 18 base pairs in length, T_(m)(° C.)=2 (# of A+T bases)+4 (# of G+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(° C.)=81.5+16.6 (log 10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na⁺] for 1×SSC=0.165 M). Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.

Negative control siRNAs should have the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate genome. Such negative controls may be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

6. To validate the effectiveness by which siRNAs destroy target mRNAs (e.g., wild-type or mutant SARS-CoV-2 mRNA), the siRNA may be incubated with target cDNA (e.g., SARS-CoV-2 cDNA) in a Drosophila-based in vitro mRNA expression system. Radiolabeled with ³²P, newly synthesized target mRNAs (e.g., SARS-CoV-2 mRNA) are detected autoradiographically on an agarose gel. The presence of cleaved target mRNA indicates mRNA nuclease activity. Suitable controls include omission of siRNA and use of non-target cDNA. Alternatively, control siRNAs are selected having the same nucleotide composition as the selected siRNA, but without significant sequence complementarity to the appropriate target gene. Such negative controls can be designed by randomly scrambling the nucleotide sequence of the selected siRNA. A homology search can be performed to ensure that the negative control lacks homology to any other gene in the appropriate genome. In addition, negative control siRNAs can be designed by introducing one or more base mismatches into the sequence.

Anti-SARS-CoV-2 siRNAs may be designed to target any of the target sequences described supra. Said siRNAs comprise an antisense strand, which is sufficiently complementary with the target sequence to mediate silencing of the target sequence. In certain embodiments, the RNA silencing agent is a siRNA.

In certain embodiments, the siRNA comprises a sense strand comprising a sequence set forth in Table 6C or Table 6D, and an antisense strand comprising a sequence set forth in Table 6B.

Sites of siRNA-mRNA complementation are selected, which result in optimal mRNA specificity and maximal mRNA cleavage.

b) siRNA-Like Molecules

siRNA-like molecules of the invention have a sequence (i.e., have a strand having a sequence) that is “sufficiently complementary” to a target sequence of an SARS-CoV-2 mRNA to direct gene silencing either by RNAi or translational repression. siRNA-like molecules are designed in the same way as siRNA molecules, but the degree of sequence identity between the sense strand and target RNA approximates that observed between a miRNA and its target. In general, as the degree of sequence identity between a miRNA sequence and the corresponding target gene sequence is decreased, the tendency to mediate post-transcriptional gene silencing by translational repression rather than RNAi is increased. Therefore, in an alternative embodiment, where post-transcriptional gene silencing by translational repression of the target gene is desired, the miRNA sequence has partial complementarity with the target gene sequence. In certain embodiments, the miRNA sequence has partial complementarity with one or more short sequences (complementarity sites) dispersed within the target mRNA (e.g. within the 3′-UTR of the target mRNA) (Hutvagner and Zamore, Science, 2002; Zeng et al., Mol. Cell, 2002; Zeng et al., RNA, 2003; Doench et al., Genes & Dev., 2003). Since the mechanism of translational repression is cooperative, multiple complementarity sites (e.g., 2, 3, 4, 5, or 6) may be targeted in certain embodiments.

The capacity of a siRNA-like duplex to mediate RNAi or translational repression may be predicted by the distribution of non-identical nucleotides between the target gene sequence and the nucleotide sequence of the silencing agent at the site of complementarity. In one embodiment, where gene silencing by translational repression is desired, at least one non-identical nucleotide is present in the central portion of the complementarity site so that duplex formed by the miRNA guide strand and the target mRNA contains a central “bulge” (Doench J G et al., Genes & Dev., 2003). In another embodiment 2, 3, 4, 5, or 6 contiguous or non-contiguous non-identical nucleotides are introduced. The non-identical nucleotide may be selected such that it forms a wobble base pair (e.g., G:U) or a mismatched base pair (G:A, C:A, C:U, G:G, A:A, C:C, U:U). In a further embodiment, the “bulge” is centered at nucleotide positions 12 and 13 from the 5′ end of the miRNA molecule.

c) Short Hairpin RNA (shRNA) Molecules

In certain featured embodiments, the instant invention provides shRNAs capable of mediating RNA silencing of an SARS-CoV-2 target sequence with enhanced selectivity. In contrast to siRNAs, shRNAs mimic the natural precursors of micro RNAs (miRNAs) and enter at the top of the gene silencing pathway. For this reason, shRNAs are believed to mediate gene silencing more efficiently by being fed through the entire natural gene silencing pathway.

miRNAs are noncoding RNAs of approximately 22 nucleotides, which can regulate gene expression at the post transcriptional or translational level during plant and animal development. One common feature of miRNAs is that they are all excised from an approximately 70 nucleotide precursor RNA stem-loop termed pre-miRNA, probably by Dicer, an RNase III-type enzyme, or a homolog thereof. Naturally-occurring miRNA precursors (pre-miRNA) have a single strand that forms a duplex stem including two portions that are generally complementary, and a loop, that connects the two portions of the stem. In typical pre-miRNAs, the stem includes one or more bulges, e.g., extra nucleotides that create a single nucleotide “loop” in one portion of the stem, and/or one or more unpaired nucleotides that create a gap in the hybridization of the two portions of the stem to each other. Short hairpin RNAs, or engineered RNA precursors, of the present application are artificial constructs based on these naturally occurring pre-miRNAs, but which are engineered to deliver desired RNA silencing agents (e.g., siRNAs of the invention). By substituting the stem sequences of the pre-miRNA with sequence complementary to the target mRNA, a shRNA is formed. The shRNA is processed by the entire gene silencing pathway of the cell, thereby efficiently mediating RNAi.

The requisite elements of a shRNA molecule include a first portion and a second portion, having sufficient complementarity to anneal or hybridize to form a duplex or double-stranded stem portion. The two portions need not be fully or perfectly complementary. The first and second “stem” portions are connected by a portion having a sequence that has insufficient sequence complementarity to anneal or hybridize to other portions of the shRNA. This latter portion is referred to as a “loop” portion in the shRNA molecule. The shRNA molecules are processed to generate siRNAs. shRNAs can also include one or more bulges, i.e., extra nucleotides that create a small nucleotide “loop” in a portion of the stem, for example a one-, two- or three-nucleotide loop. The stem portions can be the same length, or one portion can include an overhang of, for example, 1-5 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. Such Us are notably encoded by thymidines (Ts) in the shRNA-encoding DNA which signal the termination of transcription.

In shRNAs (or engineered precursor RNAs) of the instant invention, one portion of the duplex stem is a nucleic acid sequence that is complementary (or anti-sense) to the SARS-CoV-2 target sequence. In certain embodiments, one strand of the stem portion of the shRNA is sufficiently complementary (e.g., antisense) to a target RNA (e.g., mRNA) sequence to mediate degradation or cleavage of said target RNA via RNA interference (RNAi). Thus, engineered RNA precursors include a duplex stem with two portions and a loop connecting the two stem portions. The antisense portion can be on the 5′ or 3′ end of the stem. The stem portions of a shRNA are about 15 to about 50 nucleotides in length. In certain embodiments, the two stem portions are about 18 or 19 to about 21, 22, 23, 24, 25, 30, 35, 37, 38, 39, or 40 or more nucleotides in length. In certain embodiments, the length of the stem portions should be 21 nucleotides or greater. When used in mammalian cells, the length of the stem portions should be less than about 30 nucleotides to avoid provoking non-specific responses like the interferon pathway. In non-mammalian cells, the stem can be longer than 30 nucleotides. In fact, the stem can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA). In fact, a stem portion can include much larger sections complementary to the target mRNA (up to, and including the entire mRNA).

The two portions of the duplex stem must be sufficiently complementary to hybridize to form the duplex stem. Thus, the two portions can be, but need not be, fully or perfectly complementary. In addition, the two stem portions can be the same length, or one portion can include an overhang of 1, 2, 3, or 4 nucleotides. The overhanging nucleotides can include, for example, uracils (Us), e.g., all Us. The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop in the shRNAs or engineered RNA precursors can be 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length.

The loop in the shRNAs or engineered RNA precursors may differ from natural pre-miRNA sequences by modifying the loop sequence to increase or decrease the number of paired nucleotides, or replacing all or part of the loop sequence with a tetraloop or other loop sequences. Thus, the loop portion in the shRNA can be about 2 to about 20 nucleotides in length, i.e., about 2, 3, 4, 5, 6, 7, 8, 9, or more, e.g., 15 or 20, or more nucleotides in length. In certain embodiments, a loop consists of or comprises a “tetraloop” sequence. Exemplary tetraloop sequences include, but are not limited to, the sequences GNRA, where N is any nucleotide and R is a purine nucleotide, GGGG, and UUUU.

In certain embodiments, shRNAs of the present application include the sequences of a desired siRNA molecule described supra. In other embodiments, the sequence of the antisense portion of a shRNA can be designed essentially as described above or generally by selecting an 18, 19, 20, 21 nucleotides, or longer, sequence from within the target RNA (e.g., SARS-CoV-2 mRNA), for example, from a region 100 to 200 or 300 nucleotides upstream or downstream of the start of translation. In general, the sequence can be selected from any portion of the target RNA (e.g., mRNA) including the 5′ UTR (untranslated region), coding sequence, or 3′ UTR. This sequence can optionally follow immediately after a region of the target gene containing two adjacent AA nucleotides. The last two nucleotides of the nucleotide sequence can be selected to be UU. This 21 or so nucleotide sequence is used to create one portion of a duplex stem in the shRNA. This sequence can replace a stem portion of a wild-type pre-miRNA sequence, e.g., enzymatically, or is included in a complete sequence that is synthesized. For example, one can synthesize DNA oligonucleotides that encode the entire stem-loop engineered RNA precursor, or that encode just the portion to be inserted into the duplex stem of the precursor, and using restriction enzymes to build the engineered RNA precursor construct, e.g., from a wild-type pre-miRNA.

Engineered RNA precursors include, in the duplex stem, the 21-22 or so nucleotide sequences of the siRNA or siRNA-like duplex desired to be produced in vivo. Thus, the stem portion of the engineered RNA precursor includes at least 18 or 19 nucleotide pairs corresponding to the sequence of an exonic portion of the gene whose expression is to be reduced or inhibited. The two 3′ nucleotides flanking this region of the stem are chosen so as to maximize the production of the siRNA from the engineered RNA precursor and to maximize the efficacy of the resulting siRNA in targeting the corresponding mRNA for translational repression or destruction by RNAi in vivo and in vitro.

In certain embodiments, shRNAs of the invention include miRNA sequences, optionally end-modified miRNA sequences, to enhance entry into RISC. The miRNA sequence can be similar or identical to that of any naturally occurring miRNA (see e.g. The miRNA Registry; Griffiths-Jones S, Nuc. Acids Res., 2004). Over one thousand natural miRNAs have been identified to date and together they are thought to comprise about 1% of all predicted genes in the genome. Many natural miRNAs are clustered together in the introns of pre-mRNAs and can be identified in silico using homology-based searches (Pasquinelli et al., 2000; Lagos-Quintana et al., 2001; Lau et al., 2001; Lee and Ambros, 2001) or computer algorithms (e.g. MiRScan, MiRSeeker) that predict the capability of a candidate miRNA gene to form the stem loop structure of a pri-mRNA (Grad et al., Mol. Cell., 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003; Lai E C et al., Genome Bio., 2003). An online registry provides a searchable database of all published miRNA sequences (The miRNA Registry at the Sanger Institute website; Griffiths-Jones S, Nuc. Acids Res., 2004). Exemplary, natural miRNAs include lin-4, let-7, miR-10, mirR-15, miR-16, miR-168, miR-175, miR-196 and their homologs, as well as other natural miRNAs from humans and certain model organisms including Drosophila melanogaster, Caenorhabditis elegans, zebrafish, Arabidopsis thalania, Mus musculus, and Rattus norvegicus as described in International PCT Publication No. WO 03/029459.

Naturally-occurring miRNAs are expressed by endogenous genes in vivo and are processed from a hairpin or stem-loop precursor (pre-miRNA or pri-miRNAs) by Dicer or other RNAses (Lagos-Quintana et al., Science, 2001; Lau et al., Science, 2001; Lee and Ambros, Science, 2001; Lagos-Quintana et al., Curr. Biol., 2002; Mourelatos et al., Genes Dev., 2002; Reinhart et al., Science, 2002; Ambros et al., Curr. Biol., 2003; Brennecke et al., 2003; Lagos-Quintana et al., RNA, 2003; Lim et al., Genes Dev., 2003; Lim et al., Science, 2003). miRNAs can exist transiently in vivo as a double-stranded duplex, but only one strand is taken up by the RISC complex to direct gene silencing. Certain miRNAs, e.g., plant miRNAs, have perfect or near-perfect complementarity to their target mRNAs and, hence, direct cleavage of the target mRNAs. Other miRNAs have less than perfect complementarity to their target mRNAs and, hence, direct translational repression of the target mRNAs. The degree of complementarity between a miRNA and its target mRNA is believed to determine its mechanism of action. For example, perfect or near-perfect complementarity between a miRNA and its target mRNA is predictive of a cleavage mechanism (Yekta et al., Science, 2004), whereas less than perfect complementarity is predictive of a translational repression mechanism. In certain embodiments, the miRNA sequence is that of a naturally-occurring miRNA sequence, the aberrant expression or activity of which is correlated with a miRNA disorder.

d) Dual Functional Oligonucleotide Tethers

In other embodiments, the RNA silencing agents of the present invention include dual functional oligonucleotide tethers useful for the intercellular recruitment of a miRNA. Animal cells express a range of miRNAs, noncoding RNAs of approximately 22 nucleotides which can regulate gene expression at the post transcriptional or translational level. By binding a miRNA bound to RISC and recruiting it to a target mRNA, a dual functional oligonucleotide tether can repress the expression of genes involved e.g., in the arteriosclerotic process. The use of oligonucleotide tethers offers several advantages over existing techniques to repress the expression of a particular gene. First, the methods described herein allow an endogenous molecule (often present in abundance), a miRNA, to mediate RNA silencing. Accordingly, the methods described herein obviate the need to introduce foreign molecules (e.g., siRNAs) to mediate RNA silencing. Second, the RNA-silencing agents and the linking moiety (e.g., oligonucleotides such as the 2′-O-methyl oligonucleotide), can be made stable and resistant to nuclease activity. As a result, the tethers of the present invention can be designed for direct delivery, obviating the need for indirect delivery (e.g. viral) of a precursor molecule or plasmid designed to make the desired agent within the cell. Third, tethers and their respective moieties, can be designed to conform to specific mRNA sites and specific miRNAs. The designs can be cell and gene product specific. Fourth, the methods disclosed herein leave the mRNA intact, allowing one skilled in the art to block protein synthesis in short pulses using the cell's own machinery. As a result, these methods of RNA silencing are highly regulatable.

The dual functional oligonucleotide tethers (“tethers”) of the invention are designed such that they recruit miRNAs (e.g., endogenous cellular miRNAs) to a target mRNA so as to induce the modulation of a gene of interest. In certain embodiments, the tethers have the formula T-L-μ, wherein T is an mRNA targeting moiety, L is a linking moiety, and is a miRNA recruiting moiety. Any one or more moiety may be double stranded. In certain embodiments, each moiety is single stranded.

Moieties within the tethers can be arranged or linked (in the 5′ to 3′ direction) as depicted in the formula T-L-μ (i.e., the 3′ end of the targeting moiety linked to the 5′ end of the linking moiety and the 3′ end of the linking moiety linked to the 5′ end of the miRNA recruiting moiety). Alternatively, the moieties can be arranged or linked in the tether as follows: μ-T-L (i.e., the 3′ end of the miRNA recruiting moiety linked to the 5′ end of the linking moiety and the 3′ end of the linking moiety linked to the 5′ end of the targeting moiety).

The mRNA targeting moiety, as described above, is capable of capturing a specific target mRNA. According to the invention, expression of the target mRNA is undesirable, and, thus, translational repression of the mRNA is desired. The mRNA targeting moiety should be of sufficient size to effectively bind the target mRNA. The length of the targeting moiety will vary greatly, depending, in part, on the length of the target mRNA and the degree of complementarity between the target mRNA and the targeting moiety. In various embodiments, the targeting moiety is less than about 200, 100, 50, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nucleotides in length. In a certain embodiment, the targeting moiety is about 15 to about 25 nucleotides in length.

The miRNA recruiting moiety, as described above, is capable of associating with a miRNA. According to the present application, the miRNA may be any miRNA capable of repressing the target mRNA. Mammals are reported to have over 250 endogenous miRNAs (Lagos-Quintana et al. (2002) Current Biol. 12:735-739; Lagos-Quintana et al. (2001) Science 294:858-862; and Lim et al. (2003) Science 299:1540). In various embodiments, the miRNA may be any art-recognized miRNA.

The linking moiety is any agent capable of linking the targeting moieties such that the activity of the targeting moieties is maintained. Linking moieties can be oligonucleotide moieties comprising a sufficient number of nucleotides, such that the targeting agents can sufficiently interact with their respective targets. Linking moieties have little or no sequence homology with cellular mRNA or miRNA sequences. Exemplary linking moieties include one or more 2′-O-methylnucleotides, e.g., 2′-β-methyladenosine, 2′-O-methylthymidine, 2′-O-methylguanosine or 2′-O-methyluridine.

e) Gene Silencing Oligonucleotides

In certain exemplary embodiments, gene expression (i.e., SARS-CoV-2 gene expression) can be modulated using oligonucleotide-based compounds comprising two or more single stranded antisense oligonucleotides that are linked through their 5′-ends that allow the presence of two or more accessible 3′-ends to effectively inhibit or decrease SARS-CoV-2 gene expression. Such linked oligonucleotides are also known as Gene Silencing Oligonucleotides (GSOs). (See, e.g., U.S. Pat. No. 8,431,544 assigned to Idera Pharmaceuticals, Inc., incorporated herein by reference in its entirety for all purposes.)

The linkage at the 5′ ends of the GSOs is independent of the other oligonucleotide linkages and may be directly via 5′, 3′ or 2′ hydroxyl groups, or indirectly, via a non-nucleotide linker or a nucleoside, utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside. Linkages may also utilize a functionalized sugar or nucleobase of a 5′ terminal nucleotide.

GSOs can comprise two identical or different sequences conjugated at their 5′-5′ ends via a phosphodiester, phosphorothioate or non-nucleoside linker. Such compounds may comprise 15 to 27 nucleotides that are complementary to specific portions of mRNA targets of interest for antisense down regulation of a gene product. GSOs that comprise identical sequences can bind to a specific mRNA via Watson-Crick hydrogen bonding interactions and inhibit protein expression. GSOs that comprise different sequences are able to bind to two or more different regions of one or more mRNA target and inhibit protein expression. Such compounds are comprised of heteronucleotide sequences complementary to target mRNA and form stable duplex structures through Watson-Crick hydrogen bonding. Under certain conditions, GSOs containing two free 3′-ends (5′-5′-attached antisense) can be more potent inhibitors of gene expression than those containing a single free 3′-end or no free 3′-end.

In some embodiments, the non-nucleotide linker is glycerol or a glycerol homolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4 or from 1 to about 3. In some other embodiments, the non-nucleotide linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6 or from 2 to about 4.

Some non-nucleotide linkers permit attachment of more than two GSO components. For example, the non-nucleotide linker glycerol has three hydroxyl groups to which GSO components may be covalently attached. Some oligonucleotide-based compounds of the invention, therefore, comprise two or more oligonucleotides linked to a nucleotide or a non-nucleotide linker. Such oligonucleotides according to the invention are referred to as being “branched.”

In certain embodiments, GSOs are at least 14 nucleotides in length. In certain exemplary embodiments, GSOs are 15 to 40 nucleotides long or 20 to 30 nucleotides in length. Thus, the component oligonucleotides of GSOs can independently be 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides in length.

These oligonucleotides can be prepared by the art recognized methods, such as phosphoramidate or H-phosphonate chemistry, which can be carried out manually or by an automated synthesizer. These oligonucleotides may also be modified in a number of ways without compromising their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of the oligonucleotide being an alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate, carboxymethyl ester, or a combination of these and other internucleotide linkages between the 5′ end of one nucleotide and the 3′ end of another nucleotide, in which the 5′ nucleotide phosphodiester linkage has been replaced with any number of chemical groups.

V. Modified Anti-SARS-CoV-2 RNA Silencing Agents

In certain aspects of the invention, an RNA silencing agent (or any portion thereof) of the present application, as described supra, may be modified, such that the activity of the agent is further improved. For example, the RNA silencing agents described in Section II supra, may be modified with any of the modifications described infra. The modifications can, in part, serve to further enhance target discrimination, to enhance stability of the agent (e.g., to prevent degradation), to promote cellular uptake, to enhance the target efficiency, to improve efficacy in binding (e.g., to the targets), to improve patient tolerance to the agent, and/or to reduce toxicity.

1) Modifications to Enhance Target Discrimination

In certain embodiments, the RNA silencing agents of the present application may be substituted with a destabilizing nucleotide to enhance single nucleotide target discrimination (see U.S. application Ser. No. 11/698,689, filed Jan. 25, 2007 and U.S. Provisional Application No. 60/762,225 filed Jan. 25, 2006, both of which are incorporated herein by reference). Such a modification may be sufficient to abolish the specificity of the RNA silencing agent for a non-target mRNA (e.g. wild-type mRNA), without appreciably affecting the specificity of the RNA silencing agent for a target mRNA (e.g. gain-of-function mutant mRNA).

In certain embodiments, the RNA silencing agents of the present application are modified by the introduction of at least one universal nucleotide in the antisense strand thereof. Universal nucleotides comprise base portions that are capable of base pairing indiscriminately with any of the four conventional nucleotide bases (e.g. A, G, C, U). A universal nucleotide is contemplated because it has relatively minor effect on the stability of the RNA duplex or the duplex formed by the guide strand of the RNA silencing agent and the target mRNA. Exemplary universal nucleotides include those having an inosine base portion or an inosine analog base portion selected from the group consisting of deoxyinosine (e.g. 2′-deoxyinosine), 7-deaza-2′-deoxyinosine, 2′-aza-2′-deoxyinosine, PNA-inosine, morpholino-inosine, LNA-inosine, phosphoramidate-inosine, 2′-O-methoxyethyl-inosine, and 2′-OMe-inosine. In certain embodiments, the universal nucleotide is an inosine residue or a naturally occurring analog thereof.

In certain embodiments, the RNA silencing agents of the invention are modified by the introduction of at least one destabilizing nucleotide within 5 nucleotides from a specificity-determining nucleotide (i.e., the nucleotide which recognizes the disease-related polymorphism). For example, the destabilizing nucleotide may be introduced at a position that is within 5, 4, 3, 2, or 1 nucleotide(s) from a specificity-determining nucleotide. In exemplary embodiments, the destabilizing nucleotide is introduced at a position which is 3 nucleotides from the specificity-determining nucleotide (i.e., such that there are 2 stabilizing nucleotides between the destabilizing nucleotide and the specificity-determining nucleotide). In RNA silencing agents having two strands or strand portions (e.g. siRNAs and shRNAs), the destabilizing nucleotide may be introduced in the strand or strand portion that does not contain the specificity-determining nucleotide. In certain embodiments, the destabilizing nucleotide is introduced in the same strand or strand portion that contains the specificity-determining nucleotide.

2) Modifications to Enhance Efficacy and Specificity

In certain embodiments, the RNA silencing agents of the invention may be altered to facilitate enhanced efficacy and specificity in mediating RNAi according to asymmetry design rules (see U.S. Pat. Nos. 8,309,704, 7,750,144, 8,304,530, 8,329,892 and 8,309,705). Such alterations facilitate entry of the antisense strand of the siRNA (e.g., a siRNA designed using the methods of the present application or an siRNA produced from a shRNA) into RISC in favor of the sense strand, such that the antisense strand preferentially guides cleavage or translational repression of a target mRNA, and thus increasing or improving the efficiency of target cleavage and silencing. In certain embodiments, the asymmetry of an RNA silencing agent is enhanced by lessening the base pair strength between the antisense strand 5′ end (AS 5′) and the sense strand 3′ end (S 3′) of the RNA silencing agent relative to the bond strength or base pair strength between the antisense strand 3′ end (AS 3′) and the sense strand 5′ end (S ′5) of said RNA silencing agent.

In one embodiment, the asymmetry of an RNA silencing agent of the present application may be enhanced such that there are fewer G:C base pairs between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion than between the 3′ end of the first or antisense strand and the 5′ end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one mismatched base pair between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion. In certain embodiments, the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one wobble base pair, e.g., G:U, between the 5′ end of the first or antisense strand and the 3′ end of the sense strand portion. In another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a rare nucleotide, e.g., inosine (I). In certain embodiments, the base pair is selected from the group consisting of an I:A, I:U and I:C. In yet another embodiment, the asymmetry of an RNA silencing agent of the invention may be enhanced such that there is at least one base pair comprising a modified nucleotide. In certain embodiments, the modified nucleotide is selected from the group consisting of 2-amino-G, 2-amino-A, 2,6-diamino-G, and 2,6-diamino-A.

3) RNA Silencing Agents with Enhanced Stability

The RNA silencing agents of the present application can be modified to improve stability in serum or in growth medium for cell cultures. In order to enhance the stability, the 3′-residues may be stabilized against degradation, e.g., they may be selected such that they consist of purine nucleotides, such as adenosine or guanosine nucleotides. Alternatively, substitution of pyrimidine nucleotides by modified analogues, e.g., substitution of uridine by 2′-deoxythymidine is tolerated and does not affect the efficiency of RNA interference.

In a one aspect, the present application features RNA silencing agents that include first and second strands wherein the second strand and/or first strand is modified by the substitution of internal nucleotides with modified nucleotides, such that in vivo stability is enhanced as compared to a corresponding unmodified RNA silencing agent. As defined herein, an “internal” nucleotide is one occurring at any position other than the 5′ end or 3′ end of nucleic acid molecule, polynucleotide or oligonucleotide. An internal nucleotide can be within a single-stranded molecule or within a strand of a duplex or double-stranded molecule. In one embodiment, the sense strand and/or antisense strand is modified by the substitution of at least one internal nucleotide. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more internal nucleotides. In another embodiment, the sense strand and/or antisense strand is modified by the substitution of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the internal nucleotides. In yet another embodiment, the sense strand and/or antisense strand is modified by the substitution of all of the internal nucleotides.

In one aspect, the present application features RNA silencing agents that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents may be fully chemically modified, i.e., 100% of the nucleotides are chemically modified. In another aspect, the present application features RNA silencing agents comprising 2′-OH ribose groups that are at least 80% chemically modified. In certain embodiments, the RNA silencing agents comprise 2′-OH ribose groups that are about 80%, 85%, 90%, 95%, or 100% chemically modified.

In certain embodiments, the RNA silencing agents may contain at least one modified nucleotide analogue. The nucleotide analogues may be located at positions where the target-specific silencing activity, e.g., the RNAi mediating activity or translational repression activity is not substantially affected, e.g., in a region at the 5′-end and/or the 3′-end of the siRNA molecule. Moreover, the ends may be stabilized by incorporating modified nucleotide analogues.

Exemplary nucleotide analogues include sugar- and/or backbone-modified ribonucleotides (i.e., include modifications to the phosphate-sugar backbone). For example, the phosphodiester linkages of natural RNA may be modified to include at least one of a nitrogen or sulfur heteroatom. In exemplary backbone-modified ribonucleotides, the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g., of phosphorothioate group. In exemplary sugar-modified ribonucleotides, the 2′ OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.

In certain embodiments, the modifications are 2′-fluoro, 2′-amino and/or 2′-thio modifications. Modifications include 2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine, 2-amino-cytidine, 2-amino-uridine, 2-amino-adenosine, 2′-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine, and/or 5-amino-allyl-uridine. In a certain embodiment, the 2′-fluoro ribonucleotides are every uridine and cytidine. Additional exemplary modifications include 5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribothymidine, 2-aminopurine, 2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and 5-fluoro-uridine. 2′-deoxy-nucleotides and 2′-O-Me nucleotides can also be used within modified RNA-silencing agents of the instant invention. Additional modified residues include, deoxy-abasic, inosine, N3-methyl-uridine, N6,N6-dimethyl-adenosine, pseudouridine, purine ribonucleoside and ribavirin. In a certain embodiment, the 2′ moiety is a methyl group such that the linking moiety is a 2′-O-methyl oligonucleotide.

In a certain embodiment, the RNA silencing agent of the present application comprises Locked Nucleic Acids (LNAs). LNAs comprise sugar-modified nucleotides that resist nuclease activities (are highly stable) and possess single nucleotide discrimination for mRNA (Elmen et al., Nucleic Acids Res., (2005), 33(1): 439-447; Braasch et al. (2003) Biochemistry 42:7967-7975, Petersen et al. (2003) Trends Biotechnol 21:74-81). These molecules have 2′-0,4′-C-ethylene-bridged nucleic acids, with possible modifications such as 2′-deoxy-2″-fluorouridine. Moreover, LNAs increase the specificity of oligonucleotides by constraining the sugar moiety into the 3′-endo conformation, thereby pre-organizing the nucleotide for base pairing and increasing the melting temperature of the oligonucleotide by as much as 10° C. per base.

In another exemplary embodiment, the RNA silencing agent of the present application comprises Peptide Nucleic Acids (PNAs). PNAs comprise modified nucleotides in which the sugar-phosphate portion of the nucleotide is replaced with a neutral 2-amino ethylglycine moiety capable of forming a polyamide backbone, which is highly resistant to nuclease digestion and imparts improved binding specificity to the molecule (Nielsen, et al., Science, (2001), 254: 1497-1500).

Also contemplated are nucleobase-modified ribonucleotides, i.e., ribonucleotides, containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. Bases may be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and/or cytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine, 5-bromo uridine; adenosine and/or guanosines modified at the 8 position, e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O- and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable. It should be noted that the above modifications may be combined.

In other embodiments, cross-linking can be employed to alter the pharmacokinetics of the RNA silencing agent, for example, to increase half-life in the body. Thus, the present application includes RNA silencing agents having two complementary strands of nucleic acid, wherein the two strands are crosslinked. The present application also includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 3′ terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like). Modifying siRNA derivatives in this way may improve cellular uptake or enhance cellular targeting activities of the resulting siRNA derivative as compared to the corresponding siRNA, are useful for tracing the siRNA derivative in the cell, or improve the stability of the siRNA derivative compared to the corresponding siRNA.

Other exemplary modifications include: (a) 2′ modification, e.g., provision of a 2′ OMe moiety on a U in a sense or antisense strand, but especially on a sense strand, or provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); (b) modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; e.g., with the replacement of a O with an S; (c) replacement of the U with a C₅ amino linker; (d) replacement of an A with a G (sequence changes can be located on the sense strand and not the antisense strand in certain embodiments); and (d) modification at the 2′, 6′, 7′, or 8′ position. Exemplary embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications. Yet other exemplary modifications include the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a O with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrrolidone in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxen, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus.

Heavily Modified RNA Silencing Agents

In certain embodiments, the RNA silencing agent comprises at least 80% chemically modified nucleotides. In certain embodiments, the RNA silencing agent is fully chemically modified, i.e., 100% of the nucleotides are chemically modified.

In certain embodiments, the RNA silencing agent is 2′-O-methyl rich, i.e., comprises greater than 50% 2′-O-methyl content. In certain embodiments, the RNA silencing agent comprises at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 2′-O-methyl nucleotide content. In certain embodiments, the RNA silencing agent comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and sense strand. In certain embodiments, the antisense strand comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the antisense strand comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises at least about 70% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between about 70% and about 90% 2′-O-methyl nucleotide modifications. In certain embodiments, the sense strand comprises between 100% 2′-O-methyl nucleotide modifications.

2′-O-methyl rich RNA silencing agents and specific chemical modification patterns are further described in U.S. Ser. No. 16/550,076 (filed Aug. 23, 2019) and U.S. Ser. No. 62/891,185 (filed Aug. 23, 2019), each of which is incorporated herein by reference.

Internucleotide Linkage Modifications

In certain embodiments, at least one internucleotide linkage, intersubunit linkage, or nucleotide backbone is modified in the RNA silencing agent. In certain embodiments, all of the internucleotide linkages in the RNA silencing agent are modified. In certain embodiments, the modified internucleotide linkage comprises a phosphorothioate internucleotide linkage. In certain embodiments, the RNA silencing agent comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 4-16 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent comprises 8-13 phosphorothioate internucleotide linkages. In certain embodiments, the RNA silencing agent is a dsRNA comprising an antisense strand and a sense strand, each comprising a 5′ end and a 3′ end. In certain embodiments, the nucleotides at positions 1 and 2 from the 5′ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 3′ end of sense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-8 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, or 1-8 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages. In certain embodiments, the nucleotides at positions 1-2 to 1-7 from the 3′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate internucleotide linkages.

In one aspect, the disclosure provides a modified oligonucleotide, said oligonucleotide having a 5′ end, a 3′ end, that is complementary to a target, wherein the oligonucleotide comprises a sense and antisense strand, and at least one modified intersubunit linkage of Formula (I):

wherein: B is a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; X is selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, OR, NH—, NH₂, S—, and SH; Z is selected from the group consisting of O and CH₂; R is a protecting group; and

is an optional double bond.

In an embodiment of Formula (I), when W is CH,

is a double bond.

In an embodiment of Formula (I), when W selected from the group consisting of O, OCH₂, OCH, CH₂,

is a single bond.

In an embodiment of Formula (I), when Y is O—, either Z or W is not O.

In an embodiment of Formula (I), Z is CH₂ and W is CH₂. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (II):

In an embodiment of Formula (I), Z is CH₂ and W is O. In another embodiment, wherein the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (III):

In an embodiment of Formula (I), Z is O and W is CH₂. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula (IV):

In an embodiment of Formula (I), Z is O and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula V:

In an embodiment of Formula (I), Z is O and W is OCH₂. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VI:

In an embodiment of Formula (I), Z is CH₂ and W is CH. In another embodiment, the modified intersubunit linkage of Formula (I) is a modified intersubunit linkage of Formula VII:

In an embodiment of Formula (I), the base pairing moiety B is selected from the group consisting of adenine, guanine, cytosine, and uracil.

In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5′ end, a 3′ end, that is complementary to a target, wherein the siRNA comprises a sense and antisense strand, and at least one modified intersubunit linkage of any one or more of Formula (I), Formula (II), Formula (III), Formula (IV), Formula (V), Formula (VI), or Formula (VII).

In an embodiment, the modified oligonucleotide is incorporated into siRNA, said modified siRNA having a 5′ end, a 3′ end, that is complementary to a target and comprises a sense and antisense strand, wherein the siRNA comprises at least one modified intersubunit linkage is of Formula VIII:

wherein:

D is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH;

C is selected from the group consisting of O—, OH, OR¹. NH—, NH₂, S—, and SH;

A is selected from the group consisting of O and CH₂;

R¹ is a protecting group;

is an optional double bond; and the intersubunit is bridging two optionally modified nucleosides.

In an embodiment, when C is O—, either A or D is not O.

In an embodiment, D is CH₂. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (IX):

In an embodiment, D is O. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (X):

In an embodiment, D is CH₂. In another embodiment, the modified intersubunit linkage of Formula (VIII) is a modified intersubunit linkage of Formula (XI):

In an embodiment, D is CH. In another embodiment, the modified intersubunit linkage of Formula VIII is a modified intersubunit linkage of Formula (XII):

In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIV):

In an embodiment, D is OCH₂. In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XIII):

In another embodiment, the modified intersubunit linkage of Formula (VII) is a modified intersubunit linkage of Formula (XXa):

In an embodiment of the modified siRNA linkage, each optionally modified nucleoside is independently, at each occurrence, selected from the group consisting of adenosine, guanosine, cytidine, and uridine.

In certain exemplary embodiments of Formula (I), W is O. In another embodiment, W is CH₂. In yet another embodiment, W is CH.

In certain exemplary embodiments of Formula (I), X is OH. In another embodiment, X is OCH₃. In yet another embodiment, X is halo.

In a certain embodiment of Formula (I), the modified siRNA does not comprise a 2′-fluoro substituent.

In an embodiment of Formula (I), Y is O. In another embodiment, Y is OH. In yet another embodiment, Y is OR. In still another embodiment, Y is NH—. In an embodiment, Y is NH₂. In another embodiment, Y is S—. In yet another embodiment, Y is SH.

In an embodiment of Formula (I), Z is O. In another embodiment, Z is CH₂.

In an embodiment, the modified intersubunit linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the modified intersubunit linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the modified intersubunit linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the modified intersubunit linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the modified intersubunit linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), C is O—. In another embodiment, C is OH. In yet another embodiment, C is OR¹. In still another embodiment, C is NH—. In an embodiment, C is NH₂. In another embodiment, C is S—. In yet another embodiment, C is SH.

In an exemplary embodiment of the modified siRNA linkage of Formula (VIII), A is O. In another embodiment, A is CH₂. In yet another embodiment, C is OR¹. In still another embodiment, C is NH—. In an embodiment, C is NH₂. In another embodiment, C is S—. In yet another embodiment, C is SH.

In a certain embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is adenosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is guanosine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is cytidine. In another embodiment of the modified siRNA linkage of Formula (VIII), the optionally modified nucleoside is uridine.

In an embodiment of the modified siRNA linkage, wherein the linkage is inserted on position 1-2 of the antisense strand. In another embodiment, the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment, the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment, the linkage is inserted on position 19-20 of the antisense strand. In an embodiment, the linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

In certain embodiments of Formula (I), the base pairing moiety B is adenine. In certain embodiments of Formula (I), the base pairing moiety B is guanine. In certain embodiments of Formula (I), the base pairing moiety B is cytosine. In certain embodiments of Formula (I), the base pairing moiety B is uracil.

In an embodiment of Formula (I), W is O. In an embodiment of Formula (I), W is CH₂. In an embodiment of Formula (I), W is CH.

In an embodiment of Formula (I), X is OH. In an embodiment of Formula (I), X is OCH₃. In an embodiment of Formula (I), X is halo.

In an exemplary embodiment of Formula (I), the modified oligonucleotide does not comprise a 2′-fluoro substituent.

In an embodiment of Formula (I), Y is O—. In an embodiment of Formula (I), Y is OH. In an embodiment of Formula (I), Y is OR. In an embodiment of Formula (I), Y is NH—. In an embodiment of Formula (I), Y is NH₂. In an embodiment of Formula (I), Y is S—. In an embodiment of Formula (I), Y is SH.

In an embodiment of Formula (I), Z is O. In an embodiment of Formula (I), Z is CH₂.

In an embodiment of the Formula (I), the linkage is inserted on position 1-2 of the antisense strand. In another embodiment of Formula (I), the linkage is inserted on position 6-7 of the antisense strand. In yet another embodiment of Formula (I), the linkage is inserted on position 10-11 of the antisense strand. In still another embodiment of Formula (I), the linkage is inserted on position 19-20 of the antisense strand. In an embodiment of Formula (I), the linkage is inserted on positions 5-6 and 18-19 of the antisense strand.

Modified intersubunit linkages are further described in WO20200198509 and PCT/US2021/024425, each of which is incorporated herein by reference.

4) Conjugated Functional Moieties

In other embodiments, RNA silencing agents may be modified with one or more functional moieties. A functional moiety is a molecule that confers one or more additional activities to the RNA silencing agent. In certain embodiments, the functional moieties enhance cellular uptake by target cells (e.g., lung cells). Thus, the invention includes RNA silencing agents which are conjugated or unconjugated (e.g., at its 5′ and/or 3′ terminus) to another moiety (e.g. a non-nucleic acid moiety such as a peptide), an organic compound (e.g., a dye), or the like. The conjugation can be accomplished by methods known in the art, e.g., using the methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001) (describes nucleic acids loaded to polyalkylcyanoacrylate (PACA) nanoparticles); Fattal et al., J. Control Release 53 (1-3): 137-43 (1998) (describes nucleic acids bound to nanoparticles); Schwab et al., Ann. Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked to intercalating agents, hydrophobic groups, polycations or PACA nanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995) (describes nucleic acids linked to nanoparticles).

In a certain embodiment, the functional moiety is a hydrophobic moiety. In a certain embodiment, the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides and nucleoside analogs, endocannabinoids, and vitamins. In a certain embodiment, the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA). In a certain embodiment, the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA). In a certain embodiment, the vitamin selected from the group consisting of choline, vitamin A, vitamin E, and derivatives or metabolites thereof. In a certain embodiment, the vitamin is selected from the group consisting of retinoic acid and alpha-tocopheryl succinate.

In a certain embodiment, an RNA silencing agent of invention is conjugated to a lipophilic moiety. In one embodiment, the lipophilic moiety is a ligand that includes a cationic group. In another embodiment, the lipophilic moiety is attached to one or both strands of an siRNA. In an exemplary embodiment, the lipophilic moiety is attached to one end of the sense strand of the siRNA. In another exemplary embodiment, the lipophilic moiety is attached to the 3′ end of the sense strand. In certain embodiments, the lipophilic moiety is selected from the group consisting of cholesterol, vitamin E, vitamin K, vitamin A, folic acid, a cationic dye (e.g., Cy3). In an exemplary embodiment, the lipophilic moiety is cholesterol. Other lipophilic moieties include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

In certain embodiments, the functional moieties may comprise one or more ligands tethered to an RNA silencing agent to improve stability, hybridization thermodynamics with a target nucleic acid, targeting to a particular tissue or cell-type, or cell permeability, e.g., by an endocytosis-dependent or -independent mechanism. Ligands and associated modifications can also increase sequence specificity and consequently decrease off-site targeting. A tethered ligand can include one or more modified bases or sugars that can function as intercalators. These can be located in an internal region, such as in a bulge of RNA silencing agent/target duplex. The intercalator can be an aromatic, e.g., a polycyclic aromatic or heterocyclic aromatic compound. A polycyclic intercalator can have stacking capabilities, and can include systems with 2, 3, or 4 fused rings. The universal bases described herein can be included on a ligand. In one embodiment, the ligand can include a cleaving group that contributes to target gene inhibition by cleavage of the target nucleic acid. The cleaving group can be, for example, a bleomycin (e.g., bleomycin-A5, bleomycin-A2, or bleomycin-B2), pyrene, phenanthroline (e.g., 0-phenanthroline), a polyamine, a tripeptide (e.g., lys-tyr-lys tripeptide), or a metal ion chelating group. The metal ion chelating group can include, e.g., an Lu(III) or EU(III) macrocyclic complex, a Zn(II) 2,9-dimethylphenanthroline derivative, a Cu(II) terpyridine, or acridine, which can promote the selective cleavage of target RNA at the site of the bulge by free metal ions, such as Lu(III). In some embodiments, a peptide ligand can be tethered to a RNA silencing agent to promote cleavage of the target RNA, e.g., at the bulge region. For example, 1,8-dimethyl-1,3,6,8,10,13-hexaazacyclotetradecane (cyclam) can be conjugated to a peptide (e.g., by an amino acid derivative) to promote target RNA cleavage. A tethered ligand can be an aminoglycoside ligand, which can cause an RNA silencing agent to have improved hybridization properties or improved sequence specificity. Exemplary aminoglycosides include glycosylated polylysine, galactosylated polylysine, neomycin B, tobramycin, kanamycin A, and acridine conjugates of aminoglycosides, such as Neo-N-acridine, Neo-S-acridine, Neo-C-acridine, Tobra-N-acridine, and KanaA-N-acridine. Use of an acridine analog can increase sequence specificity. For example, neomycin B has a high affinity for RNA as compared to DNA, but low sequence-specificity. An acridine analog, neo-5-acridine, has an increased affinity for the HIV Rev-response element (RRE). In some embodiments, the guanidine analog (the guanidinoglycoside) of an aminoglycoside ligand is tethered to an RNA silencing agent. In a guanidinoglycoside, the amine group on the amino acid is exchanged for a guanidine group. Attachment of a guanidine analog can enhance cell permeability of an RNA silencing agent. A tethered ligand can be a poly-arginine peptide, peptoid or peptidomimetic, which can enhance the cellular uptake of an oligonucleotide agent.

Exemplary ligands are coupled, either directly or indirectly, via an intervening tether, to a ligand-conjugated carrier. In certain embodiments, the coupling is through a covalent bond. In certain embodiments, the ligand is attached to the carrier via an intervening tether. In certain embodiments, a ligand alters the distribution, targeting or lifetime of an RNA silencing agent into which it is incorporated. In certain embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand.

Exemplary ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified RNA silencing agent, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides. Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; nuclease-resistance conferring moieties; and natural or unusual nucleobases. General examples include lipophiles, lipids, steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g., triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin A, biotin, pyridoxal), carbohydrates, proteins, protein binding agents, integrin targeting molecules, polycationics, peptides, polyamines, and peptide mimics. Ligands can include a naturally occurring substance, (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); amino acid, or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine (GalNAc) or derivatives thereof, N-acetyl-glucosamine, multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. Other examples of ligands include dyes, intercalating agents (e.g. acridines and substituted acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine, phenanthroline, pyrenes), lys-tyr-lys tripeptide, aminoglycosides, guanidium aminoglycosides, artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol (and thio analogs thereof), cholic acid, cholanic acid, lithocholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, glycerol (e.g., esters (e.g., mono, bis, or tris fatty acid esters, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, Cis, C₁₉, or C₂₀ fatty acids) and ethers thereof, e.g., C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, or C₂₀ alkyl; e.g., 1,3-bis-O(hexadecyl)glycerol, 1,3-bis-O(octaadecyl)glycerol), geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, stearic acid (e.g., glyceryl distearate), oleic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, naproxen, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu³⁺ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP. In certain embodiments, the ligand is GalNAc or a derivative thereof.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the RNA silencing agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin. The ligand can increase the uptake of the RNA silencing agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNF D), interleukin-1 beta, or gamma interferon. In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can bind a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA. A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. In a certain embodiment, the lipid based ligand binds HSA. A lipid-based ligand can bind HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is contemplated that the affinity not be so strong that the HSA-ligand binding cannot be reversed. In another embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These can be useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent can be an alpha-helical agent, which may have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the RNA silencing agent, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long. A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. The peptide moiety can be an L-peptide or D-peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature 354:82-84, 1991). In exemplary embodiments, the peptide or peptidomimetic tethered to an RNA silencing agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of an antisense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 5′ end and/or 3′ end of a sense strand of the RNA silencing agent of the disclosure. In certain embodiments, the functional moiety is linked to the 3′ end of a sense strand of the RNA silencing agent of the disclosure.

In certain embodiments, the functional moiety is linked to the RNA silencing agent by a linker. In certain embodiments, the functional moiety is linked to the antisense strand and/or sense strand by a linker. In certain embodiments, the functional moiety is linked to the 3′ end of a sense strand by a linker. In certain embodiments, the linker comprises a divalent or trivalent linker. In certain embodiments, the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof. In certain embodiments, the divalent or trivalent linker is selected from:

wherein n is 1, 2, 3, 4, or 5.

In certain embodiments, the linker further comprises a phosphodiester or phosphodiester derivative. In certain embodiments, the phosphodiester or phosphodiester derivative is selected from the group consisting of.

wherein X is O, S or BH₃.

The various functional moieties of the disclosure and means to conjugate them to RNA silencing agents are described in further detail in WO2017/030973A1 and WO2018/031933A2, incorporated herein by reference.

VI. Branched Oligonucleotides

Two or more RNA silencing agents as disclosed supra, for example oligonucleotide constructs such as anti-SARS-CoV-2 siRNAs, may be connected to one another by one or more moieties independently selected from a linker, a spacer and a branching point, to form a branched oligonucleotide RNA silencing agent. In certain embodiments, the branched oligonucleotide RNA silencing agent consists of two siRNAs to form a di-branched siRNA (“di-siRNA”) scaffolding for delivering two siRNAs. In representative embodiments, the nucleic acids of the branched oligonucleotide each comprise an antisense strand (or portions thereof), wherein the antisense strand has sufficient complementarity to a target mRNA (e.g., SARS-CoV-2 mRNA) to mediate an RNA-mediated silencing mechanism (e.g. RNAi).

In exemplary embodiments, the branched oligonucleotides may have two to eight RNA silencing agents attached through a linker. The linker may be hydrophobic. In an embodiment, branched oligonucleotides of the present application have two to three oligonucleotides. In an embodiment, the oligonucleotides independently have substantial chemical stabilization (e.g., at least 40% of the constituent bases are chemically-modified). In an exemplary embodiment, the oligonucleotides have full chemical stabilization (i.e., all the constituent bases are chemically-modified). In some embodiments, branched oligonucleotides comprise one or more single-stranded phosphorothioated tails, each independently having two to twenty nucleotides. In a non-limiting embodiment, each single-stranded tail has two to ten nucleotides.

In certain embodiments, branched oligonucleotides are characterized by three properties: (1) a branched structure, (2) full metabolic stabilization, and (3) the presence of a single-stranded tail comprising phosphorothioate linkers. In certain embodiments, branched oligonucleotides have 2 or 3 branches. It is believed that the increased overall size of the branched structures promotes increased uptake. Also, without being bound by a particular theory of activity, multiple adjacent branches (e.g., 2 or 3) are believed to allow each branch to act cooperatively and thus dramatically enhance rates of internalization, trafficking and release.

Branched oligonucleotides are provided in various structurally diverse embodiments. In some embodiments nucleic acids attached at the branching points are single stranded or double stranded and consist of miRNA inhibitors, gapmers, mixmers, SSOs, PMOs, or PNAs. These single strands can be attached at their 3′ or 5′ end. Combinations of siRNA and single stranded oligonucleotides could also be used for dual function. In another embodiment, short nucleic acids complementary to the gapmers, mixmers, miRNA inhibitors, SSOs, PMOs, and PNAs are used to carry these active single-stranded nucleic acids and enhance distribution and cellular internalization. The short duplex region has a low melting temperature (T_(m) ˜37° C.) for fast dissociation upon internalization of the branched structure into the cell.

The Di-siRNA branched oligonucleotides may comprise chemically diverse conjugates, such as the functional moieties described above. Conjugated bioactive ligands may be used to enhance cellular specificity and to promote membrane association, internalization, and serum protein binding. Examples of bioactive moieties to be used for conjugation include DHA, GalNAc, and cholesterol. These moieties can be attached to Di-siRNA either through the connecting linker or spacer, or added via an additional linker or spacer attached to another free siRNA end.

Branched oligonucleotides comprise a variety of therapeutic nucleic acids, including siRNAs, ASOs, miRNAs, miRNA inhibitors, splice switching, PMOs, PNAs. In some embodiments, branched oligonucleotides further comprise conjugated hydrophobic moieties and exhibit unprecedented silencing and efficacy in vitro and in vivo.

Linkers

In an embodiment of the branched oligonucleotide, each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. In one embodiment, each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment, each linker is a peptide. In another embodiment, each linker is RNA. In another embodiment, each linker is DNA. In another embodiment, each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment, each linker is a phosphoramidate. In another embodiment, each linker is an ester. In another embodiment, each linker is an amide. In another embodiment, each linker is a triazole.

VII. Compound of Formula (I)

In another aspect, provided herein is a branched oligonucleotide compound of formula (I):

L-(N)_(n)  (I)

wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein formula (I) optionally further comprises one or more branch point B, and one or more spacer S; wherein B is independently for each occurrence a polyvalent organic species or derivative thereof, S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof.

Moiety N is an RNA duplex comprising a sense strand and an antisense strand; and n is 2, 3, 4, 5, 6, 7 or 8. In an embodiment, the antisense strand of N comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10, as recited in Table 4 and Table 5.

In an embodiment, the antisense strand of N comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions recited in Table 6A.

The sense strand and antisense strand may each independently comprise one or more chemical modifications.

In an embodiment, the compound of formula (I) has a structure selected from formulas (I-1)-(I-9) of Table 1.

TABLE 1

(I-1)

(I-2)

(I-3)

(I-4)

(I-5)

(I-6)

(I-7)

(I-8)

(I-9)

In one embodiment, the compound of formula (I) is formula (I-1). In another embodiment, the compound of formula (I) is formula (I-2). In another embodiment, the compound of formula (I) is formula (I-3). In another embodiment, the compound of formula (I) is formula (I-4). In another embodiment, the compound of formula (I) is formula (I-5). In another embodiment, the compound of formula (I) is formula (I-6). In another embodiment, the compound of formula (I) is formula (I-7). In another embodiment, the compound of formula (I) is formula (I-8). In another embodiment, the compound of formula (I) is formula (I-9).

In an embodiment of the compound of formula (I), each linker is independently selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; wherein any carbon or oxygen atom of the linker is optionally replaced with a nitrogen atom, bears a hydroxyl substituent, or bears an oxo substituent. In one embodiment of the compound of formula (I), each linker is an ethylene glycol chain. In another embodiment, each linker is an alkyl chain. In another embodiment of the compound of formula (I), each linker is a peptide. In another embodiment of the compound of formula (I), each linker is RNA. In another embodiment of the compound of formula (I), each linker is DNA. In another embodiment of the compound of formula (I), each linker is a phosphate. In another embodiment, each linker is a phosphonate. In another embodiment of the compound of formula (I), each linker is a phosphoramidate. In another embodiment of the compound of formula (I), each linker is an ester. In another embodiment of the compound of formula (I), each linker is an amide. In another embodiment of the compound of formula (I), each linker is a triazole.

In one embodiment of the compound of formula (I), B is a polyvalent organic species. In another embodiment of the compound of formula (I), B is a derivative of a polyvalent organic species. In one embodiment of the compound of formula (I), B is a triol or tetrol derivative. In another embodiment, B is a tri- or tetra-carboxylic acid derivative. In another embodiment, B is an amine derivative. In another embodiment, B is a tri- or tetra-amine derivative. In another embodiment, B is an amino acid derivative. In another embodiment of the compound of formula (I), B is selected from the formulas of.

Polyvalent organic species are moieties comprising carbon and three or more valencies (i.e., points of attachment with moieties such as S, L or N, as defined above). Non-limiting examples of polyvalent organic species include triols (e.g., glycerol, phloroglucinol, and the like), tetrols (e.g., ribose, pentaerythritol, 1,2,3,5-tetrahydroxybenzene, and the like), tri-carboxylic acids (e.g., citric acid, 1,3,5-cyclohexanetricarboxylic acid, trimesic acid, and the like), tetra-carboxylic acids (e.g., ethylenediaminetetraacetic acid, pyromellitic acid, and the like), tertiary amines (e.g., tripropargylamine, triethanolamine, and the like), triamines (e.g., diethylenetriamine and the like), tetramines, and species comprising a combination of hydroxyl, thiol, amino, and/or carboxyl moieties (e.g., amino acids such as lysine, serine, cysteine, and the like).

In an embodiment of the compound of formula (I), each nucleic acid comprises one or more chemically-modified nucleotides. In an embodiment of the compound of formula (I), each nucleic acid consists of chemically-modified nucleotides. In certain embodiments of the compound of formula (I), >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of each nucleic acid comprises chemically-modified nucleotides.

In an embodiment, each antisense strand independently comprises a 5′ terminal group R selected from the groups of Table 2.

TABLE 2

R¹

R²

R³

R⁴

R⁵

R⁶

R⁷

R⁸

In one embodiment, R is R₁. In another embodiment, R is R₂. In another embodiment, R is R₃. In another embodiment, R is R₄. In another embodiment, R is R₅. In another embodiment, R is R₆. In another embodiment, R is R₇. In another embodiment, R is R₈.

Structure of Formula (II)

In an embodiment, the compound of formula (I) has the structure of formula (II):

wherein X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage; = represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

In certain embodiments, the structure of formula (II) does not contain mismatches. In one embodiment, the structure of formula (II) contains 1 mismatch. In another embodiment, the compound of formula (II) contains 2 mismatches. In another embodiment, the compound of formula (II) contains 3 mismatches. In another embodiment, the compound of formula (II) contains 4 mismatches. In an embodiment, each nucleic acid consists of chemically-modified nucleotides.

In certain embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (II) are chemically-modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (II) are chemically-modified nucleotides.

Structure of Formula (III)

In an embodiment, the compound of formula (I) has the structure of formula (III):

wherein X, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification; Y, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; and Y, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification.

In an embodiment, X is chosen from the group consisting of 2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, X is chosen from the group consisting of 2′-O-methyl modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2′-O-methyl modified adenosine, guanosine, uridine or cytidine.

In certain embodiments, the structure of formula (III) does not contain mismatches. In one embodiment, the structure of formula (III) contains 1 mismatch. In another embodiment, the compound of formula (III) contains 2 mismatches. In another embodiment, the compound of formula (III) contains 3 mismatches. In another embodiment, the compound of formula (III) contains 4 mismatches.

Structure of Formula (IV)

In an embodiment, the compound of formula (I) has the structure of formula (IV):

wherein X, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; Y, for each occurrence, independently, is selected from adenosine, guanosine, uridine, cytidine, and chemically-modified derivatives thereof; - represents a phosphodiester internucleoside linkage; =represents a phosphorothioate internucleoside linkage; and --- represents, individually for each occurrence, a base-pairing interaction or a mismatch.

In certain embodiments, the structure of formula (IV) does not contain mismatches. In one embodiment, the structure of formula (IV) contains 1 mismatch. In another embodiment, the compound of formula (IV) contains 2 mismatches. In another embodiment, the compound of formula (IV) contains 3 mismatches. In another embodiment, the compound of formula (IV) contains 4 mismatches. In an embodiment, each nucleic acid consists of chemically-modified nucleotides.

In certain embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (IV) are chemically-modified nucleotides. In other embodiments, >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >55% or >50% of X's of the structure of formula (IV) are chemically-modified nucleotides.

Structure of Formula (V)

In an embodiment, the compound of formula (I) has the structure of formula (V):

wherein X, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; X, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification; Y, for each occurrence, independently, is a nucleotide comprising a 2′-deoxy-2′-fluoro modification; and Y, for each occurrence, independently, is a nucleotide comprising a 2′-O-methyl modification.

In certain embodiments, X is chosen from the group consisting of 2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, X is chosen from the group consisting of 2′-O-methyl modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2′-deoxy-2′-fluoro modified adenosine, guanosine, uridine or cytidine. In an embodiment, Y is chosen from the group consisting of 2′-O-methyl modified adenosine, guanosine, uridine or cytidine.

In certain embodiments, the structure of formula (V) does not contain mismatches. In one embodiment, the structure of formula (V) contains 1 mismatch. In another embodiment, the compound of formula (V) contains 2 mismatches. In another embodiment, the compound of formula (V) contains 3 mismatches. In another embodiment, the compound of formula (V) contains 4 mismatches.

Variable Linkers

In an embodiment of the compound of formula (I), L has the structure of L1:

In an embodiment of L1, R is R³ and n is 2.

In an embodiment of the structure of formula (II), L has the structure of L1. In an embodiment of the structure of formula (III), L has the structure of L1. In an embodiment of the structure of formula (IV), L has the structure of L1. In an embodiment of the structure of formula (V), L has the structure of L1. In an embodiment of the structure of formula (VI), L has the structure of L1. In an embodiment of the structure of formula (VI), L has the structure of L1.

In an embodiment of the compound of formula (I), L has the structure of L2:

In an embodiment of L2, R is R3 and n is 2. In an embodiment of the structure of formula (II), L has the structure of L2. In an embodiment of the structure of formula (III), L has the structure of L2. In an embodiment of the structure of formula (IV), L has the structure of L2. In an embodiment of the structure of formula (V), L has the structure of L2. In an embodiment of the structure of formula (VI), L has the structure of L2. In an embodiment of the structure of formula (VI), L has the structure of L2.

Delivery System

In a third aspect, provided herein is a delivery system for therapeutic nucleic acids having the structure of formula (VI):

L-(cNA)_(n)  (VI)

wherein L is selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof, wherein formula (VI) optionally further comprises one or more branch point B, and one or more spacer S; wherein B is independently for each occurrence a polyvalent organic species or derivative thereof; S is independently for each occurrence selected from an ethylene glycol chain, an alkyl chain, a peptide, RNA, DNA, a phosphate, a phosphonate, a phosphoramidate, an ester, an amide, a triazole, and combinations thereof; each cNA, independently, is a carrier nucleic acid comprising one or more chemical modifications; and n is 2, 3, 4, 5, 6, 7 or 8.

In one embodiment of the delivery system, L is an ethylene glycol chain. In another embodiment of the delivery system, L is an alkyl chain. In another embodiment of the delivery system, L is a peptide. In another embodiment of the delivery system, L is RNA. In another embodiment of the delivery system, L is DNA. In another embodiment of the delivery system, L is a phosphate. In another embodiment of the delivery system, L is a phosphonate. In another embodiment of the delivery system, L is a phosphoramidate. In another embodiment of the delivery system, L is an ester. In another embodiment of the delivery system, L is an amide. In another embodiment of the delivery system, L is a triazole.

In one embodiment of the delivery system, S is an ethylene glycol chain. In another embodiment, S is an alkyl chain. In another embodiment of the delivery system, S is a peptide. In another embodiment, S is RNA. In another embodiment of the delivery system, S is DNA. In another embodiment of the delivery system, S is a phosphate. In another embodiment of the delivery system, S is a phosphonate. In another embodiment of the delivery system, S is a phosphoramidate. In another embodiment of the delivery system, S is an ester. In another embodiment, S is an amide. In another embodiment, S is a triazole.

In one embodiment of the delivery system, n is 2. In another embodiment of the delivery system, n is 3. In another embodiment of the delivery system, n is 4. In another embodiment of the delivery system, n is 5. In another embodiment of the delivery system, n is 6. In another embodiment of the delivery system, n is 7. In another embodiment of the delivery system, n is 8.

In certain embodiments, each cNA comprises >95%, >90%, >85%, >80%, >75%, >70%, >65%, >60%, >5500 or >5000 chemically-modified nucleotides.

In an embodiment, the compound of formula (VI) has a structure selected from formulas (VI-1)-(VI-9) of Table 3:

TABLE 3

(VI-1)

(VI-2)

(VI-3)

(VI-4)

(VI-5)

(VI-6)

(VI-7)

(VI-8)

(VI-9)

In an embodiment, the compound of formula (VI) is the structure of formula (VI-1). In an embodiment, the compound of formula (VI) is the structure of formula (VI-2). In an embodiment, the compound of formula (VI) is the structure of formula (VI-3). In an embodiment, the compound of formula (VI) is the structure of formula (VI-4). In an embodiment, the compound of formula (VI) is the structure of formula (VI-5). In an embodiment, the compound of formula (VI) is the structure of formula (VI-6). In an embodiment, the compound of formula (VI) is the structure of formula (VI-7). In an embodiment, the compound of formula (VI) is the structure of formula (VI-8). In an embodiment, the compound of formula (VI) is the structure of formula (VI-9).

In an embodiment, the compound of formulas (VI) (including, e.g., formulas (VI-1)-(VI-9), each cNA independently comprises at least 15 contiguous nucleotides. In an embodiment, each cNA independently consists of chemically-modified nucleotides.

In an embodiment, the delivery system further comprises n therapeutic nucleic acids (NA), wherein each NA comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10, as recited in Table 4 and Table. In further embodiments, NA includes strands that are capable of targeting one or more SARS-CoV-2 nucleic acid sequences of any one of the 45-nucleotide target gene regions recited in Table 6A.

Also, each NA is hybridized to at least one cNA. In one embodiment, the delivery system is comprised of 2 NAs. In another embodiment, the delivery system is comprised of 3 NAs. In another embodiment, the delivery system is comprised of 4 NAs. In another embodiment, the delivery system is comprised of 5 NAs. In another embodiment, the delivery system is comprised of 6 NAs. In another embodiment, the delivery system is comprised of 7 NAs. In another embodiment, the delivery system is comprised of 8 NAs.

In an embodiment, each NA independently comprises at least 15 contiguous nucleotides. In an embodiment, each NA independently comprises 15-25 contiguous nucleotides. In an embodiment, each NA independently comprises 15 contiguous nucleotides. In an embodiment, each NA independently comprises 16 contiguous nucleotides. In another embodiment, each NA independently comprises 17 contiguous nucleotides. In another embodiment, each NA independently comprises 18 contiguous nucleotides. In another embodiment, each NA independently comprises 19 contiguous nucleotides. In another embodiment, each NA independently comprises 20 contiguous nucleotides. In an embodiment, each NA independently comprises 21 contiguous nucleotides. In an embodiment, each NA independently comprises 22 contiguous nucleotides. In an embodiment, each NA independently comprises 23 contiguous nucleotides. In an embodiment, each NA independently comprises 24 contiguous nucleotides. In an embodiment, each NA independently comprises 25 contiguous nucleotides.

In an embodiment, each NA comprises an unpaired overhang of at least 2 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 3 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 4 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 5 nucleotides. In another embodiment, each NA comprises an unpaired overhang of at least 6 nucleotides. In an embodiment, the nucleotides of the overhang are connected via phosphorothioate linkages.

In an embodiment, each NA, independently, is selected from the group consisting of: DNA, siRNAs, antagomiRs, miRNAs, gapmers, mixmers, or guide RNAs. In one embodiment, each NA, independently, is a DNA. In another embodiment, each NA, independently, is a siRNA. In another embodiment, each NA, independently, is an antagomiR. In another embodiment, each NA, independently, is a miRNA. In another embodiment, each NA, independently, is a gapmer. In another embodiment, each NA, independently, is a mixmer. In another embodiment, each NA, independently, is a guide RNA. In an embodiment, each NA is the same. In an embodiment, each NA is not the same.

In an embodiment, the delivery system further comprising n therapeutic nucleic acids (NA) has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein. In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 2 therapeutic nucleic acids (NA). In another embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 3 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 4 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 5 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 6 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 7 therapeutic nucleic acids (NA). In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), and embodiments thereof described herein further comprising 8 therapeutic nucleic acids (NA).

In one embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), further comprising a linker of structure L1 or L2 wherein R is R³ and n is 2. In another embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), further comprising a linker of structure L1 wherein R is R³ and n is 2. In another embodiment, the delivery system has a structure selected from formulas (I), (II), (III), (IV), (V), (VI), further comprising a linker of structure L2 wherein R is R³ and n is 2.

In an embodiment of the delivery system, the target of delivery is selected from the group consisting of: lung, brain, liver, skin, kidney, spleen, pancreas, colon, fat, muscle, adrenal glands, and thymus. In one embodiment, the target of delivery is the lung. In another embodiment, the target of delivery are alveolar cells in the lung. In another embodiment, the target of delivery are club cells in the lung. In another embodiment, the target of delivery is the striatum of the brain. In one embodiment, the target of delivery is the liver. In one embodiment, the target of delivery is the skin. In one embodiment, the target of delivery is the kidney. In one embodiment, the target of delivery is the spleen. In one embodiment, the target of delivery is the pancreas. In one embodiment, the target of delivery is the colon. In one embodiment, the target of delivery is the fat. In one embodiment, the target of delivery are the adrenal glands. In one embodiment, the target of delivery is the muscle. In one embodiment, the target of delivery is the thymus. In one embodiment, the target of delivery is the spinal cord.

In one embodiment, efficacy of delivery to lung cells is achieved through combinations of unique conjugates, optimization of siRNA stability, structural configuration, cleavable linker, and Phosphorothioate (PS) content. In another embodiment, three conjugates, EPA, DCA and PC-DCA, have different distribution profiles. In one embodiment, DCA and PC-DCA are being cleared mostly by the liver and EPA is being cleared mostly by the kidneys. In another embodiment the two classes of conjugates show different cell-type preferences in the lung, where EPA accumulation is higher in epithelial (Clara) cells of the lung.

In certain embodiments, compounds of the invention are characterized by the following properties: (1) two or more branched oligonucleotides, e.g., wherein there is a non-equal number of 3′ and 5′ ends; (2) substantially chemically stabilized, e.g., wherein more than 40%, optimally 100%, of oligonucleotides are chemically modified (e.g., no RNA and optionally no DNA); and (3) phoshorothioated single oligonucleotides containing at least 3, phosphorothioated bonds. In certain embodiments, the phoshorothioated single oligonucleotides contain 4-20 phosphorothioated bonds.

It is to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein; as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by M R Green and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

Branched oligonucleotides, including synthesis and methods of use, are described in greater detail in WO2017/132669, incorporated herein by reference.

Methods of Introducing Nucleic Acids, Vectors and Host Cells

RNA silencing agents of the invention may be directly introduced into the cell (e.g., a cell in the lung) (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

The RNA silencing agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The nucleic acid may be introduced along with other components that perform one or more of the following activities: enhance nucleic acid uptake by the cell or other-wise increase inhibition of the target gene.

Physical methods of introducing nucleic acids include injection of a solution containing the RNA, bombardment by particles covered by the RNA, soaking the cell or organism in a solution of the RNA, or electroporation of cell membranes in the presence of the RNA. A viral construct packaged into a viral particle would accomplish both efficient introduction of an expression construct into the cell and transcription of RNA encoded by the expression construct. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, such as calcium phosphate, and the like. Thus, the RNA may be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, inhibit annealing of single strands, stabilize the single strands, or other-wise increase inhibition of the target gene.

RNA may be directly introduced into the cell (i.e., intracellularly); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the RNA. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the RNA may be introduced.

The cell having the target gene may be from the germ line or somatic, totipotent or pluripotent, dividing or non-dividing, parenchyma or epithelium, immortalized or transformed, or the like. The cell may be a stem cell or a differentiated cell. Cell types that are differentiated include adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium, neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages, neutrophils, eosinophils, basophils, mast cells, leukocytes, granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts, hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double stranded RNA material delivered, this process may provide partial or complete loss of function for the target gene. A reduction or loss of gene expression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more of targeted cells is exemplary. Inhibition of gene expression refers to the absence (or observable decrease) in the level of protein and/or mRNA product from a target gene. Specificity refers to the ability to inhibit the target gene without manifest effects on other genes of the cell. The consequences of inhibition can be confirmed by examination of the outward properties of the cell or organism (as presented below in the examples) or by biochemical techniques such as RNA solution hybridization, nuclease protection, Northern hybridization, reverse transcription, gene expression monitoring with a microarray, antibody binding, Enzyme Linked ImmunoSorbent Assay (ELISA), Western blotting, RadioImmunoAssay (RIA), other immunoassays, and Fluorescence Activated Cell Sorting (FACS).

For RNA-mediated inhibition in a cell line or whole organism, gene expression is conveniently assayed by use of a reporter or drug resistance gene whose protein product is easily assayed. Such reporter genes include acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase (NOS), octopine synthase (OCS), and derivatives thereof. Multiple selectable markers are available that confer resistance to ampicillin, bleomycin, chloramphenicol, gentamycin, hygromycin, kanamycin, lincomycin, methotrexate, phosphinothricin, puromycin, and tetracycline. Depending on the assay, quantitation of the amount of gene expression allows one to determine a degree of inhibition which is greater than 10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treated according to the present invention. Lower doses of injected material and longer times after administration of RNAi agent may result in inhibition in a smaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or 95% of targeted cells). Quantization of gene expression in a cell may show similar amounts of inhibition at the level of accumulation of target mRNA or translation of target protein. As an example, the efficiency of inhibition may be determined by assessing the amount of gene product in the cell; mRNA may be detected with a hybridization probe having a nucleotide sequence outside the region used for the inhibitory double-stranded RNA, or translated polypeptide may be detected with an antibody raised against the polypeptide sequence of that region.

The RNA may be introduced in an amount which allows delivery of at least one copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per cell) of material may yield more effective inhibition; lower doses may also be useful for specific applications.

In an exemplary aspect, the efficacy of an RNAi agent of the invention (e.g., an siRNA targeting an SARS-CoV-2 target sequence) is tested for its ability to specifically degrade mutant mRNA (e.g., SARS-CoV-2 mRNA and/or the production of SARS-CoV-2 protein) in cells, such as cells in the lung. In certain embodiments, cells in the lung include, but are not limited to, clara cells, alveolar cells, and club cells. Also suitable for cell-based validation assays are other readily transfectable cells, for example, HeLa cells or COS cells. Cells are transfected with human wild type or mutant cDNAs (e.g., human wild type or mutant SARS-CoV-2 cDNA). Standard siRNA, modified siRNA or vectors able to produce siRNA from U-looped mRNA are co-transfected. Selective reduction in target mRNA (e.g., SARS-CoV-2 mRNA) and/or target protein (e.g., SARS-CoV-2 protein) is measured. Reduction of target mRNA or protein can be compared to levels of target mRNA or protein in the absence of an RNAi agent or in the presence of an RNAi agent that does not target SARS-CoV-2 mRNA. Exogenously-introduced mRNA or protein (or endogenous mRNA or protein) can be assayed for comparison purposes. When utilizing lung cells, it may be desirable to introduce RNAi agents (e.g., siRNAs) by passive uptake.

Recombinant Adeno-Associated Viruses and Vectors

In certain exemplary embodiments, recombinant adeno-associated viruses (rAAVs) and their associated vectors can be used to deliver one or more siRNAs into cells, e.g., lung cells (e.g., clara cells, alveolar cells or club cells). AAV is able to infect many different cell types, although the infection efficiency varies based upon serotype, which is determined by the sequence of the capsid protein. Several native AAV serotypes have been identified, with serotypes 1-9 being the most commonly used for recombinant AAV. AAV-2 is the most well-studied and published serotype. The AAV-DJ system includes serotypes AAV-DJ and AAV-DJ/8. These serotypes were created through DNA shuffling of multiple AAV serotypes to produce AAV with hybrid capsids that have improved transduction efficiencies in vitro (AAV-DJ) and in vivo (AAV-DJ/8) in a variety of cells and tissues.

In certain embodiments, widespread lung delivery can be achieved by intratracheal (IT) delivery of recombinant adeno-associated virus 7 (rAAV7), RAAV9 and rAAV10, or other suitable rAAVs. rAAVs and their associated vectors are well-known in the art and are described in US Patent Applications 2014/0296486, 2010/0186103, 2008/0269149, 2006/0078542 and 2005/0220766, each of which is incorporated herein by reference in its entirety for all purposes.

rAAVs may be delivered to a subject in compositions according to any appropriate methods known in the art. An rAAV can be suspended in a physiologically compatible carrier (i.e., in a composition), and may be administered to a subject, i.e., a host animal, such as a human, mouse, rat, cat, dog, sheep, rabbit, horse, cow, goat, pig, guinea pig, hamster, chicken, turkey, a non-human primate (e.g., Macaque) or the like. In certain embodiments, a host animal is a non-human host animal.

Delivery of one or more rAAVs to a mammalian subject may be performed, for example, by intramuscular injection or by administration into the bloodstream of the mammalian subject. Administration into the bloodstream may be by injection into a vein, an artery, or any other vascular conduit. In certain embodiments, one or more rAAVs are administered into the bloodstream by way of isolated limb perfusion, a technique well known in the surgical arts, the method essentially enabling the artisan to isolate a limb from the systemic circulation prior to administration of the rAAV virions. A variant of the isolated limb perfusion technique, described in U.S. Pat. No. 6,177,403, can also be employed by the skilled artisan to administer virions into the vasculature of an isolated limb to potentially enhance transduction into muscle cells or tissue. Moreover, in certain instances, it may be desirable to deliver virions to the lung of a subject. By “lung” is meant all cells and tissue of the lung of a vertebrate. Thus, the term includes, but is not limited to, clara cells, alveolar cells, club cells, and the like. Recombinant AAVs may be delivered directly to the lung by injection, e.g., intratracheal injection.

The compositions of the invention may comprise an rAAV alone, or in combination with one or more other viruses (e.g., a second rAAV encoding having one or more different transgenes). In certain embodiments, a composition comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different rAAVs each having one or more different transgenes.

An effective amount of an rAAV is an amount sufficient to target infect an animal, target a desired tissue. In some embodiments, an effective amount of an rAAV is an amount sufficient to produce a stable somatic transgenic animal model. The effective amount will depend primarily on factors such as the species, age, weight, health of the subject, and the tissue to be targeted, and may thus vary among animal and tissue. For example, an effective amount of one or more rAAVs is generally in the range of from about 1 ml to about 100 ml of solution containing from about 109 to 10¹⁶ genome copies. In some cases, a dosage between about 10¹¹ to 10¹² rAAV genome copies is appropriate. In certain embodiments, 10¹² rAAV genome copies is effective to target lung, heart, liver, and pancreas tissues. In some cases, stable transgenic animals are produced by multiple doses of an rAAV.

In some embodiments, rAAV compositions are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., about 10¹³ genome copies/mL or more). Methods for reducing aggregation of rAAVs are well known in the art and, include, for example, addition of surfactants, pH adjustment, salt concentration adjustment, etc. (See, e.g., Wright et al. (2005) Molecular Therapy 12:171-178, the contents of which are incorporated herein by reference.)

“Recombinant AAV (rAAV) vectors” comprise, at a minimum, a transgene and its regulatory sequences, and 5′ and 3′ AAV inverted terminal repeats (ITRs). It is this recombinant AAV vector which is packaged into a capsid protein and delivered to a selected target cell. In some embodiments, the transgene is a nucleic acid sequence, heterologous to the vector sequences, which encodes a polypeptide, protein, functional RNA molecule (e.g., siRNA) or other gene product, of interest. The nucleic acid coding sequence is operatively linked to regulatory components in a manner which permits transgene transcription, translation, and/or expression in a cell of a target tissue.

The AAV sequences of the vector typically comprise the cis-acting 5′ and 3′ inverted terminal repeat (ITR) sequences (See, e.g., B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990)). The ITR sequences are usually about 145 basepairs in length. In certain embodiments, substantially the entire sequences encoding the ITRs are used in the molecule, although some degree of minor modification of these sequences is permissible. The ability to modify these ITR sequences is within the skill of the art. (See, e.g., texts such as Sambrook et al, “Molecular Cloning. A Laboratory Manual”, 2d ed., Cold Spring Harbor Laboratory, New York (1989); and K. Fisher et al., J Virol., 70:520 532 (1996)). An example of such a molecule employed in the present invention is a “cis-acting” plasmid containing the transgene, in which the selected transgene sequence and associated regulatory elements are flanked by the 5′ and 3′ AAV ITR sequences. The AAV ITR sequences may be obtained from any known AAV, including mammalian AAV types described further herein.

VIII. Methods of Treatment

In one aspect, the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) SARS-CoV-2 infection. In one embodiment, the disease or disorder is such that SARS-CoV-2 levels in blood or another biological sample have been found to be a marker of infection. In another embodiment, the infection with SARS-CoV-2 is characterized by a clinical manifestation of viral infection, e.g. an increase in body temperature. In a certain embodiment, a reduction in SARS-CoV-2 mRNA reduces clinical manifestations of SARS-CoV-2 infection.

“Treatment,” or “treating,” as used herein, is defined as the application or administration of a therapeutic agent (e.g., a RNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

In one aspect, the invention provides a method for preventing in a subject, a disease or disorder as described above, by administering to the subject a therapeutic agent (e.g., an RNAi agent or vector or transgene encoding same). Subjects at risk for the disease can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the disease or disorder, such that the disease or disorder is prevented or, alternatively, delayed in its progression.

Another aspect of the invention pertains to methods treating subjects therapeutically, i.e., alter onset of symptoms of the disease or disorder. In an exemplary embodiment, the modulatory method of the invention involves contacting a lung cell with a therapeutic agent (e.g., a RNAi agent or vector or transgene encoding same) that is specific for a target sequence within the gene (e.g., SARS-CoV-2 target sequences of Tables 4, 5, 6A, 7, and 8), such that sequence specific interference with the gene is achieved. These methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).

IX. Pharmaceutical Compositions and Methods of Administration

The invention pertains to uses of the above-described agents for prophylactic and/or therapeutic treatments as described infra. Accordingly, the modulators (e.g., RNAi agents) of the present invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, antibody, or modulatory compound and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation), transdermal (topical), and transmucosal administration.

The nucleic acid molecules of the invention can be inserted into expression constructs, e.g., viral vectors, retroviral vectors, expression cassettes, or plasmid viral vectors, e.g., using methods known in the art, including but not limited to those described in Xia et al., (2002), Supra. Expression constructs can be delivered to a subject by, for example, inhalation, orally, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994), Proc. Natl. Acad. Sci. USA, 91, 3054-3057). The pharmaceutical preparation of the delivery vector can include the vector in an acceptable diluent, or can comprise a slow release matrix in which the delivery vehicle is imbedded. Alternatively, where the complete delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

The nucleic acid molecules of the invention can also include small hairpin RNAs (shRNAs), and expression constructs engineered to express shRNAs. Transcription of shRNAs is initiated at a polymerase III (pol III) promoter, and is thought to be terminated at position 2 of a 4-5-thymine transcription termination site. Upon expression, shRNAs are thought to fold into a stem-loop structure with 3′ UU-overhangs; subsequently, the ends of these shRNAs are processed, converting the shRNAs into siRNA-like molecules of about 21 nucleotides. Brummelkamp et al. (2002), Science, 296, 550-553; Lee et al, (2002). supra; Miyagishi and Taira (2002), Nature Biotechnol., 20, 497-500; Paddison et al. (2002), supra; Paul (2002), supra; Sui (2002) supra; Yu et al. (2002), supra.

The expression constructs may be any construct suitable for use in the appropriate expression system and include, but are not limited to retroviral vectors, linear expression cassettes, plasmids and viral or virally-derived vectors, as known in the art. Such expression constructs may include one or more inducible promoters, RNA Pol III promoter systems such as U6 snRNA promoters or H1 RNA polymerase III promoters, or other promoters known in the art. The constructs can include one or both strands of the siRNA. Expression constructs expressing both strands can also include loop structures linking both strands, or each strand can be separately transcribed from separate promoters within the same construct. Each strand can also be transcribed from a separate expression construct, Tuschl (2002), Supra.

In certain embodiments, a composition that includes a compound of the invention can be delivered to the lungs of a subject by a variety of routes. Exemplary routes include intratracheal or nasal delivery. The composition can also be delivered systemically, e.g., by intravenous, subcutaneous or intramuscular injection.

For example, compositions can include one or more species of a compound of the invention and a pharmaceutically acceptable carrier. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated.

As used herein “intratracheal administration” refers to the direct administration to the lung through the trachea. Intratracheal administration includes, but is not limited to, intratracheal inhalation and intratracheal instillation. Intratracheal administration (IT) is non-invasive and can be used in the field. Both the chemical architectures of the siRNAs and routes of administration might have benefits in different clinical contexts and disease stages. The chemical architectures optimal for different routes of administration is different. For example, DCA-conjugated siRNAs or divalent siRNAs can be effective for delivery of siRNA to the lung.

In one embodiment, divalent siRNAs are delivered to lung tissues. A variety of lung delivery systems can be employed to accomplish delivery to the lung tissues, for example, but not limited to, direct intrathecal instillation, or by using a nebulizer. Formulations in which the siRNA can be delivered are, for example, but not limited to, dry powder, direct powder, vapor droplets, etc.

It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following example, which is included for purposes of illustration only and is not intended to be limiting.

EXAMPLES Example 1. In Vitro Identification of SARS-CoV-2 Targeting Sequences

Hyper functional siRNAs targeting all 9 genes (regions) of SARS-CoV2 were identified. A combination of bioinformatic approaches were employed to identify regions of conservation (based on 718 patients isolates) in combination with features essential for RISC entry and tolerance of chemical modifications. Over 100 chemically optimized compounds were synthesized, and reporter systems developed to test these compounds in cells, screened at 1.5 μM concentration. Selected hits were further screened in dose response studies, and at least two lead compounds were identified per gene with IC₅₀ values <20 nM.

Target sequences were derived from the severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) isolate Wuhan-Hu-1 (NCBI accession: NC_045512). Fully chemically modified and conjugated oligonucleotides targeting SARS-CoV-2 and the human host receptors of SARS-CoV2 could potentially prevent and treat viral infections from viruses within the family Coronaviridiae.

Nine SARS-CoV-2 transcripts were selected for knockdown: the genes coding for the four major structural proteins spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), and nucleocapsid protein (N), as well as genes coding for ppla, pplab, which make up the 16 non-structural proteins of SARS-CoV-2, and genes coding for the accessory proteins 3a, 8b, 7a (FIG. 1 ). Table 4 shows the full-length sequence of the SARS-CoV-2 genome, Table 5 the sequences of the SARS-CoV-2 genes. FIG. 2 depicts a diagram of siRNA and antisense oligonucleotides (ASOs) target positions on encoded proteins in the SARS-CoV-2 genome. siRNAs were designed to target nine genes encoding SARS-CoV-2 proteins: orfla, orflab, spike surface glycoprotein (S), small envelope protein (E), matrix protein (M), nucleocapsid protein (N), and accessory proteins 3a, 8b, 7a. Grey arrows indicate siRNA and ASO target positions. Inset in FIG. 2 shows a detailed view of siRNA target positions on genes in the 3′ region of the genome.

TABLE 4 SARS-CoV2 genomic sequence (NCBI Accession Number NC 045512) SARS-CoV2 genome sequence ATTAAAGGTTTATACCTTCCCAGGTAACAAACCAACCAACTTTCGATCTC TTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTGTGGCTGTCACTC GGCTGCATGCTTAGTGCACTCACGCAGTATAATTAATAACTAATTACTGT CGTTGACAGGACACGAGTAACTCGTCTATCTTCTGCAGGCTGCTTACGGT TTCGTCCGTGTTGCAGCCGATCATCAGCACATCTAGGTTTCGTCCGGGTG TGACCGAAAGGTAAGATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAAC ACACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGACGTGCTCGTAC GTGGCTTTGGAGACTCCGTGGAGGAGGTCTTATCAGAGGCACGTCAACAT CTTAAAGATGGCACTTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCC TCAACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGATGCTCGAACTG CACCTCATGGTCATGTTATGGTTGAGCTGGTAGCAGAACTCGAAGGCATT CAGTACGGTCGTAGTGGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGG CGAAATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGAACGGTAATA AAGGAGCTGGTGGCCATAGTTACGGCGCCGATCTAAAGTCATTTGACTTA GGCGACGAGCTTGGCACTGATCCTTATGAAGATTTTCAAGAAAACTGGAA CACTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCGTGAGCTTAACG GAGGGGCATACACTCGCTATGTCGATAACAACTTCTGTGGCCCTGATGGC TACCCTCTTGAGTGCATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTC ATGCACTTTGTCCGAACAACTGGACTTTATTGACACTAAGAGGGGTGTAT ACTGCTGCCGTGAACATGAGCATGAAATTGCTTGGTACACGGAACGTTCT GAAAAGAGCTATGAATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAA ATTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATTTCCCTTAAATT CCATAATCAAGACTATTCAACCAAGGGTTGAAAAGAAAAAGCTTGATGGC TTTATGGGTAGAATTCGATCTGTCTATCCAGTTGCGTCACCAAATGAATG CAACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCATTGTGGTGAAA CTTCATGGCAGACGGGCGATTTTGTTAAAGCCACTTGCGAATTTTGTGGC ACTGAGAATTTGACTAAAGAAGGTGCCACTACTTGTGGTTACTTACCCCA AAATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAATTCAGAAGTAG GACCTGAGCATAGTCTTGCCGAATACCATAATGAATCTGGCTTGAAAACC ATTCTTCGTAAGGGTGGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTC TTATGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCACGTGCTAGCG CTAACATAGGTTGTAACCATACAGGTGTTGTTGGAGAAGGTTCCGAAGGT CTTAATGACAACCTTCTTGAAATACTCCAAAAAGAGAAAGTCAACATCAA TATTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATTATTTTGGCAT CTTTTTCTGCTTCCACAAGTGCTTTTGTGGAAACTGTGAAAGGTTTGGAT TATAAAGCATTCAAACAAATTGTTGAATCCTGTGGTAATTTTAAAGTTAC AAAAGGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAACAGAAATCAA TACTGAGTCCTCTTTATGCATTTGCATCAGAGGCTGCTCGTGTTGTACGA TCAATTTTCTCCCGCACTCTTGAAACTGCTCAAAATTCTGTGCGTGTTTT ACAGAAGGCCGCTATAACAATACTAGATGGAATTTCACAGTATTCACTGA GACTCATTGATGCTATGATGTTCACATCTGATTTGGCTACTAACAATCTA GTTGTAATGGCCTACATTACAGGTGGTGTTGTTCAGTTGACTTCGCAGTG GCTAACTAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCGTCCTTG ATTGGCTTGAAGAGAAGTTTAAGGAAGGTGTAGAGTTTCTTAGAGACGGT TGGGAAATTGTTAAATTTATCTCAACCTGTGCTTGTGAAATTGTCGGTGG ACAAATTGTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGACATTCT TTAAGCTTGTAAATAAATTTTTGGCTTTGTGTGCTGACTCTATCATTATT GGTGGAGCTAAACTTAAAGCCTTGAATTTAGGTGAAACATTTGTCACGCA CTCAAAGGGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAACTGGCC TACTCATGCCTCTAAAAGCCCCAAAAGAAATTATCTTCTTAGAGGGAGAA ACACTTCCCACAGAAGTGTTAACAGAGGAAGTTGTCTTGAAAACTGGTGA TTTACAACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGCTCCATTGG TTGGTACACCAGTTTGTATTAACGGGCTTATGTTGCTCGAAATCAAAGAC ACAGAAAAGTACTGTGCCCTTGCACCTAATATGATGGTAACAAACAATAC CTTCACACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGGTGATGACA CTGTGATAGAAGTGCAAGGTTACAAGAGTGTGAATATCACTTTTGAACTT GATGAAAGGATTGATAAAGTACTTAATGAGAAGTGCTCTGCCTATACAGT TGAACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTGGCAGATGCTG TCATAAAAACTTTGCAACCAGTATCTGAATTACTTACACCACTGGGCATT GATTTAGATGAGTGGAGTATGGCTACATACTACTTATTTGATGAGTCTGG TGAGTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCCTCCAGATG AGGATGAAGAAGAAGGTGATTGTGAAGAAGAAGAGTTTGAGCCATCAACT CAATATGAGTATGGTACTGAAGATGATTACCAAGGTAAACCTTTGGAATT TGGTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCAAGAAGAAGATT GGTTAGATGATGATAGTCAACAAACTGTTGGTCAACAAGACGGCAGTGAG GACAATCAGACAACTACTATTCAAACAATTGTTGAGGTTCAACCTCAATT AGAGATGGAACTTACACCAGTTGTTCAGACTATTGAAGTGAATAGTTTTA GTGGTTATTTAAAACTTACTGACAATGTATACATTAAAAATGCAGACATT GTGGAAGAAGCTAAAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAA TGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCCTTAAATAAGGCTA CTAACAATGCCATGCAAGTTGAATCTGATGATTACATAGCTACTAATGGA CCACTTAAAGTGGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTAA ACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAAGGTGAAGACATTC AACTTCTTAAGAGTGCTTATGAAAATTTTAATCAGCACGAAGTTCTACTT GCACCATTATTATCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTT AAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACTTAGCTGTCTTTG ATAAAAATCTCTATGACAAACTTGTTTCAAGCTTTTTGGAAATGAAGAGT GAAAAGCAAGTTGAACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAA GCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAGAGAAAACAAGATG ATAAGAAAATCAAAGCTTGTGTTGAAGAAGTTACAACAACTCTGGAAGAA ACTAAGTTCCTCACAGAAAACTTGTTACTTTATATTGACATTAATGGCAA TCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATTGACATCACTTTCT TAAAGAAAGATGCTCCATATATAGTGGGTGATGTTGTTCAAGAGGGTGTT TTAACTGCTGTGGTTATACCTACTAAAAAGGCTGGTGGCACTACTGAAAT GCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAATTATATAACCACTT ACCCGGGTCAGGGTTTAAATGGTTACACTGTAGAGGAGGCAAAGACAGTG CTTAAAAAGTGTAAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAA TGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATTTGCGAGAAATGC TTGCACATGCAGAAGAAACACGCAAATTAATGCCTGTCTGTGTGGAAACT AAAGCCATAGTTTCAACTATACAGCGTAAATATAAGGGTATTAAAATACA AGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTTACACCAGTAAAA CAACTGTAGCGTCACTTATCAACACACTTAACGATCTAAATGAAACTCTT GTTACAATGCCACTTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGC TGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACAGTTTCTGTTTCTT CACCTGATGCTGTTACAGCGTATAATGGTTATCTTACTTCTTCTTCTAAA ACACCTGAAGAACATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAA AGATTGGTCCTATTCTGGACAATCTACACAACTAGGTATAGAATTTCTTA AGAGAGGTGATAAAAGTGTATATTACACTAGTAATCCTACCACATTCCAC CTAGATGGTGAAGTTATCACCTTTGACAATCTTAAGACACTTCTTTCTTT GAGAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGACAACATTAACC TCCACACGCAAGTTGTGGACATGTCAATGACATATGGACAACAGTTTGGT CCAACTTATTTGGATGGAGCTGATGTTACTAAAATAAAACCTCATAATTC ACATGAAGGTAAAACATTTTATGTTTTACCTAATGATGACACTCTACGTG TTGAGGCTTTTGAGTACTACCACACAACTGATCCTAGTTTTCTGGGTAGG TACATGTCAGCATTAAATCACACTAAAAAGTGGAAATACCCACAAGTTAA TGGTTTAACTTCTATTAAATGGGCAGATAACAACTGTTATCTTGCCACTG CATTGTTAACACTCCAACAAATAGAGTTGAAGTTTAATCCACCTGCTCTA CAAGATGCTTATTACAGAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGC ACTTATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTAGGTGATGTTA GAGAAACAATGAGTTACTTGTTTCAACATGCCAATTTAGATTCTTGCAAA AGAGTCTTGAACGTGGTGTGTAAAACTTGTGGACAACAGCAGACAACCCT TAAGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCTTATGAACAAT TTAAGAAAGGTGTTCAGATACCTTGTACGTGTGGTAAACAAGCTACAAAA TATCTAGTACAACAGGAGTCACCTTTTGTTATGATGTCAGCACCACCTGC TCAGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGTGAGTACACTG GTAATTACCAGTGTGGTCACTATAAACATATAACTTCTAAAGAAACTTTG TATTGCATAGACGGTGCTTTACTTACAAAGTCCTCAGAATACAAAGGTCC TATTACGGATGTTTTCTACAAAGAAAACAGTTACACAACAACCATAAAAC CAGTTACTTATAAATTGGATGGTGTTGTTTGTACAGAAATTGACCCTAAG TTGGACAATTATTATAAGAAAGACAATTCTTATTTCACAGAGCAACCAAT TGATCTTGTACCAAACCAACCATATCCAAACGCAAGCTTCGATAATTTTA AGTTTGTATGTGATAATATCAAATTTGCTGATGATTTAAACCAGTTAACT GGTTATAAGAAACCTGCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGA CTTAAATGGTGATGTGGTGGCTATTGATTATAAACACTACACACCCTCTT TTAAGAAAGGAGCTAAATTGTTACATAAACCTATTGTTTGGCATGTTAAC AATGCAACTAATAAAGCCACGTATAAACCAAATACCTGGTGTATACGTTG TCTTTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTTGATGTACTGA AGTCAGAGGACGCGCAGGGAATGGATAATCTTGCCTGCGAAGATCTAAAA CCAGTCTCTGAAGAAGTAGTGGAAAATCCTACCATACAGAAAGACGTTCT TGAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACATTATACTTAAAC CAGCAAATAATAGTTTAAAAATTACAGAAGAGGTTGGCCACACAGATCTA ATGGCTGCTTATGTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGA ATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTCATGGTTTAGCTG CTGTTAATAGTGTCCCTTGGGATACTATAGCTAATTATGCTAAGCCTTTT CTTAACAAAGTTGTTAGTACAACTACTAACATAGTTACACGGTGTTTAAA CCGTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTATTGCTACAAT TGTGTACTTTTACTAGAAGTACAAATTCTAGAATTAAAGCATCTATGCCG ACTACTATAGCAAAGAATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGA GGCTTCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACTGATAAATA TTATAATTTGGTTTTTACTATTAAGTGTTTGCCTAGGTTCTTTAATCTAC TCAACCGCTGCTTTAGGTGTTTTAATGTCTAATTTAGGCATGCCTTCTTA CTGTACTGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCACTATTG CAACCTACTGTACTGGTTCTATACCTTGTAGTGTTTGTCTTAGTGGTTTA GATTCTTTAGACACCTATCCTTCTTTAGAAACTATACAAATTACCATTTC ATCTTTTAAATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGTTTT TGGCATATATTCTTTTCACTAGGTTTTTCTATGTACTTGGATTGGCTGCA ATCATGCAATTGTTTTTCAGCTATTTTGCAGTACATTTTATTAGTAATTC TTGGCTTATGTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAG CTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATTATGTATGGAAA AGTTATGTGCATGTTGTAGACGGTTGTAATTCATCAACTTGTATGATGTG TTACAAACGTAATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAATG GTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAAAGGCTTTTGC AAACTACACAATTGGAATTGTGTTAATTGTGATACATTCTGTGCTGGTAG TACATTTATTAGTGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAAA GACCAATAAATCCTACTGACCAGTCTTCTTACATCGTTGATAGTGTTACA GTGAAGAATGGTTCCATCCATCTTTACTTTGATAAAGCTGGTCAAAAGAC TTATGAAAGACATTCTCTCTCTCATTTTGTTAACTTAGACAACCTGAGAG CTAATAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTTTTGATGGT AAATCAAAATGTGAAGAATCATCTGCAAAATCAGCGTCTGTTTACTACAG TCAGCTTATGTGTCAACCTATACTGTTACTAGATCAGGCATTAGTGTCTG ATGTTGGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATGCTTACGTT AATACGTTTTCATCAACTTTTAACGTACCAATGGAAAAACTCAAAACACT AGTTGCAACTGCAGAAGCTGAACTTGCAAAGAATGTGTCCTTAGACAATG TCTTATCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGATTCAGAT GTAGAAACTAAAGATGTTGTTGAATGTCTTAAATTGTCACATCAATCTGA CATAGAAGTTACTGGCGATAGTTGTAATAACTATATGCTCACCTATAACA AAGTTGAAAACATGACACCCCGTGACCTTGGTGCTTGTATTGACTGTAGT GCGCGTCATATTAATGCGCAGGTAGCAAAAAGTCACAACATTGCTTTGAT ATGGAACGTTAAAGATTTCATGTCATTGTCTGAACAACTACGAAAACAAA TACGTAGTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACATGTGCA ACTACTAGACAAGTTGTTAATGTTGTAACAACAAAGATAGCACTTAAGGG TGGTAAAATTGTTAATAATTGGTTGAAGCAGTTAATTAAAGTTACACTTG TGTTCCTTTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCATGTC ATGTCTAAACATACTGACTTTTCAAGTGAAATCATAGGATACAAGGCTAT TGATGGTGGTGTCACTCGTGACATAGCATCTACAGATACTTGTTTTGCTA ACAAACATGCTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGTTAT ACTAATGACAAAGCTTGCCCATTGATTGCTGCAGTCATAACAAGAGAAGT GGGTTTTGTCGTGCCTGGTTTGCCTGGCACGATATTACGCACAACTAATG GTGACTTTTTGCATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACATC TGTTACACACCATCAAAACTTATAGAGTACACTGACTTTGCAACATCAGC TTGTGTTTTGGCTGCTGAATGTACAATTTTTAAAGATGCTTCTGGTAAGC CAGTACCATATTGTTATGATACCAATGTACTAGAAGGTTCTGTTGCTTAT GAAAGTTTACGCCCTGACACACGTTATGTGCTCATGGATGGCTCTATTAT TCAATTTCCTAACACCTACCTTGAAGGTTCTGTTAGAGTGGTAACAACTT TTGATTCTGAGTACTGTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGT GTTTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATGATTATTACAG ATCTTTACCAGGAGTTTTCTGTGGTGTAGATGCTGTAAATTTACTTACTA ATATGTTTACACCACTAATTCAACCTATTGGTGCTTTGGACATATCAGCA TCTATAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGCCTTGCCTA CTATTTTATGAGGTTTAGAAGAGCTTTTGGTGAATACAGTCATGTAGTTG CCTTTAATACTTTACTATTCCTTATGTCATTCACTGTACTCTGTTTAACA CCAGTTTACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGTACTT GACATTTTATCTTACTAATGATGTTTCTTTTTTAGCACATATTCAGTGGA TGGTTATGTTCACACCTTTAGTACCTTTCTGGATAACAATTGCTTATATC ATTTGTATTTCCACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAA GAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTTTTGAAGAAGCTG CGCTGTGCACCTTTTTGTTAAATAAAGAAATGTATCTAAAGTTGCGTAGT GATGTGCTATTACCTCTTACGCAATATAATAGATACTTAGCTCTTTATAA TAAGTACAAGTATTTTAGTGGAGCAATGGATACAACTAGCTACAGAGAAG CTGCTTGTTGTCATCTCGCAAAGGCTCTCAATGACTTCAGTAACTCAGGT TCTGATGTTCTTTACCAACCACCACAAACCTCTATCACCTCAGCTGTTTT GCAGAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAAGTTGAGGGTT GTATGGTACAAGTAACTTGTGGTACAACTACACTTAACGGTCTTTGGCTT GATGACGTAGTTTACTGTCCAAGACATGTGATCTGCACCTCTGAAGACAT GCTTAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTAATCATAATT TCTTGGTACAGGCTGGTAATGTTCAACTCAGGGTTATTGGACATTCTATG CAAAATTGTGTACTTAAGCTTAAGGTTGATACAGCCAATCCTAAGACACC TAAGTATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTCAGTGTTAG CTTGTTACAATGGTTCACCATCTGGTGTTTACCAATGTGCTATGAGGCCC AATTTCACTATTAAGGGTTCATTCCTTAATGGTTCATGTGGTAGTGTTGG TTTTAACATAGATTATGACTGTGTCTCTTTTTGTTACATGCACCATATGG AATTACCAACTGGAGTTCATGCTGGCACAGACTTAGAAGGTAACTTTTAT GGACCTTTTGTTGACAGGCAAACAGCACAAGCAGCTGGTACGGACACAAC TATTACAGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAAATGGAG ACAGGTGGTTTCTCAATCGATTTACCACAACTCTTAATGACTTTAACCTT GTGGCTATGAAGTACAATTATGAACCTCTAACACAAGACCATGTTGACAT ACTAGGACCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATATGTGTG CTTCATTAAAAGAATTACTGCAAAATGGTATGAATGGACGTACCATATTG GGTAGTGCTTTATTAGAAGATGAATTTACACCTTTTGATGTTGTTAGACA ATGCTCAGGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCAAGGGTA CACACCACTGGTTGTTACTCACAATTTTGACTTCACTTTTAGTTTTAGTC CAGAGTACTCAATGGTCTTTGTTCTTTTTTTTGTATGAAAATGCCTTTTT ACCTTTTGCTATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGTTTG TCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGTTACCTTCTCTTGCC ACTGTAGCTTATTTTAATATGGTCTATATGCCTGCTAGTTGGGTGATGCG TATTATGACATGGTTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGC TAAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCCTTATG ACAGCAAGAACTGTGTATGATGATGGTGCTAGGAGAGTGTGGACACTTAT GAATGTCTTGACACTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATC AAGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTTCTAACTACTCA GGTGTAGTTACAACTGTCATGTTTTTGGCCAGAGGTATTGTTTTTATGTG TGTTGAGTATTGCCCTATTTTCTTCATAACTGGTAATACACTTCAGTGTA TAATGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTTACTTTGGC CTCTTTTGTTTACTCAACCGCTACTTTAGACTGACTCTTGGTGTTTATGA TTACTTAGTTTCTACACAGGAGTTTAGATATATGAATTCACAGGGACTAC TCCCACCCAAGAATAGCATAGATGCCTTCAAACTCAACATTAAATTGTTG GGTGTTGGTGGCAAACCTTGTATCAAAGTAGCCACTGTACAGTCTAAAAT GTCAGATGTAAAGTGCACATCAGTAGTCTTACTCTCAGTTTTGCAACAAC TCAGAGTAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAGTTACAC AATGACATTCTCTTAGCTAAAGATACTACTGAAGCCTTTGAAAAAATGGT TTCACTACTTTCTGTTTTGCTTTCCATGCAGGGTGCTGTAGACATAAACA AGCTTTGTGAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTATAGCC TCAGAGTTTAGTTCCCTTCCATCATATGCAGCTTTTGCTACTGCTCAAGA AGCTTATGAGCAGGCTGTTGCTAATGGTGATTCTGAAGTTGTTCTTAAAA AGTTGAAGAAGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGATGCA GCCATGCAACGTAAGTTGGAAAAGATGGCTGATCAAGCTATGACCCAAAT GTATAAACAGGCTAGATCTGAGGACAAGAGGGCAAAAGTTACTAGTGCTA TGCAGACAATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGCACTC AACAACATTATCAACAATGCAAGAGATGGTTGTGTTCCCTTGAACATAAT ACCTCTTACAACAGCAGCCAAACTAATGGTTGTCATACCAGACTATAACA CATATAAAAATACGTGTGATGGTACAACATTTACTTATGCATCAGCATTG TGGGAAATCCAACAGGTTGTAGATGCAGATAGTAAAATTGTTCAACTTAG TGAAATTAGTATGGACAATTCACCTAATTTAGCATGGCCTCTTATTGTAA CAGCTTTAAGGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCTTAGT CCTGTTGCACTACGACAGATGTCTTGTGCTGCCGGTACTACACAAACTGC TTGCACTGATGACAATGCGTTAGCTTACTACAACACAACAAAGGGAGGTA GGTTTGTACTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGCTAGA TTCCCTAAGAGTGATGGAACTGGTACTATCTATACAGAACTGGAACCACC TTGTAGGTTTGTTACAGACACACCTAAAGGTCCTAAAGTGAAGTATTTAT ACTTTATTAAAGGATTAAACAACCTAAATAGAGGTATGGTACTTGGTAGT TTAGCTGCCACAGTACGTCTACAAGCTGGTAATGCAACAGAAGTGCCTGC CAATTCAACTGTATTATCTTTCTGTGCTTTTGCTGTAGATGCTGCTAAAG CTTACAAAGATTATCTAGCTAGTGGGGGACAACCAATCACTAATTGTGTT AAGATGTTGTGTACACACACTGGTACTGGTCAGGCAATAACAGTTACACC GGAAGCCAATATGGATCAAGAATCCTTTGGTGGTGCATCGTGTTGTCTGT ACTGCCGTTGCCACATAGATCATCCAAATCCTAAAGGATTTTGTGACTTA AAAGGTAAGTATGTACAAATACCTACAACTTGTGCTAATGACCCTGTGGG TTTTACACTTAAAAACACAGTCTGTACCGTCTGCGGTATGTGGAAAGGTT ATGGCTGTAGTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCTGAT GCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGTGCAGCCCGTCTTACA CCGTGCGGCACAGGCACTAGTACTGATGTCGTATACAGGGCTTTTGACAT CTACAATGATAAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTGTT GTCGCTTCCAAGAAAAGGACGAAGATGACAATTTAATTGATTCTTACTTT GTAGTTAAGAGACACACTTTCTCTAACTACCAACATGAAGAAACAATTTA TAATTTACTTAAGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAGT TTAGAATAGACGGTGACATGGTACCACATATATCACGTCAACGTCTTACT AAATACACAATGGCAGACCTCGTCTATGCTTTAAGGCATTTTGATGAAGG TAATTGTGACACATTAAAAGAAATACTTGTCACATACAATTGTTGTGATG ATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGAT ATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTT AAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTG TACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGT GATTTCATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTA TTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAG AGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTG TTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTT TAAATATTGGGATCAGACATACCACCCAAATTGTGTTAACTGTTTGGATG ACAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTATTCTCTACAGTG TTCCCACCTACAAGTTTTGGACCACTAGTGAGAAAAATATTTGTTGATGG TGTTCCATTTGTAGTTTCAACTGGATACCACTTCAGAGAGCTAGGTGTTG TACATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGTTTTAAGGAA TTACTTGTGTATGCTGCTGACCCTGCTATGCACGCTGCTTCTGGTAATCT ATTACTAGATAAACGCACTACGTGCTTTTCAGTAGCTGCACTTACTAACA ATGTTGCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGACTTCTAT GACTTTGCTGTGTCTAAGGGTTTCTTTAAGGAAGGAAGTTCTGTTGAATT AAAACACTTCTTCTTTGCTCAGGATGGTAATGCTGCTATCAGCGATTATG ACTACTATCGTTATAATCTACCAACAATGTGTGATATCAGACAACTACTA TTTGTAGTTGAAGTTGTTGATAAGTACTTTGATTGTTACGATGGTGGCTG TATTAATGCTAACCAAGTCATCGTCAACAACCTAGACAAATCAGCTGGTT TTCCATTTAATAAATGGGGTAAGGCTAGACTTTATTATGATTCAATGAGT TATGAGGATCAAGATGCACTTTTCGCATATACAAAACGTAATGTCATCCC TACTATAACTCAAATGAATCTTAAGTATGCCATTAGTGCAAAGAATAGAG CTCGCACCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAATAGACAG TTTCATCAAAAATTATTGAAATCAATAGCCGCCACTAGAGGAGCTACTGT AGTAATTGGAACAAGCAAATTCTATGGTGGTTGGCACAACATGTTAAAAA CTGTTTATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGATTATCCT AAATGTGATAGAGCCATGCCTAACATGCTTAGAATTATGGCCTCACTTGT TCTTGCTCGCAAACATACAACGTGTTGTAGCTTGTCACACCGTTTCTATA GATTAGCTAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATGTGTGGC GGTTCACTATATGTTAAACCAGGTGGAACCTCATCAGGAGATGCCACAAC TGCTTATGCTAATAGTGTTTTTAACATTTGTCAAGCTGTCACGGCCAATG TTAATGCACTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTATGTC CGCAATTTACAACACAGACTTTATGAGTGTCTCTATAGAAATAGAGATGT TGACACAGACTTTGTGAATGAGTTTTACGCATATTTGCGTAAACATTTCT CAATGATGATACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACTTAT GCATCTCAAGGTCTAGTGGCTAGCATAAAGAACTTTAAGTCAGTTCTTTA TTATCAAAACAATGTTTTTATGTCTGAAGCAAAATGTTGGACTGAGACTG ACCTTACTAAAGGACCTCATGAATTTTGCTCTCAACATACAATGCTAGTT AAACAGGGTGATGATTATGTGTACCTTCCTTACCCAGATCCATCAAGAAT CCTAGGGGCCGGCTGTTTTGTAGATGATATCGTAAAAACAGATGGTACAC TTATGATTGAACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTTACT AAACATCCTAATCAGGAGTATGCTGATGTCTTTCATTTGTACTTACAATA CATAAGAAAGCTACATGATGAGTTAACAGGACACATGTTAGACATGTATT CTGTTATGCTTACTAATGATAACACTTCAAGGTATTGGGAACCTGAGTTT TATGAGGCTATGTACACACCGCATACAGTCTTACAGGCTGTTGGGGCTTG TGTTCTTTGCAATTCACAGACTTCATTAAGATGTGGTGCTTGCATACGTA GACCATTCTTATGTTGTAAATGCTGTTACGACCATGTCATATCAACATCA CATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCAATGCTCCAGGTTG TGATGTCACAGATGTGACTCAACTTTACTTAGGAGGTATGAGCTATTATT GTAAATCACATAAACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAA GTTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGATAATGTTACTGA CTTTAATGCAATTGCAACATGTGACTGGACAAATGCTGGTGATTACATTT TAGCTAACACCTGTACTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTC AAAGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTGCTACTGTACG TGAAGTGCTGTCTGACAGAGAATTACATCTTTCATGGGAAGTTGGTAAAC CTAGACCACCACTTAACCGAAATTATGTCTTTACTGGTTATCGTGTAACT AAAAACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAAAAGGTGACTA TGGTGATGCTGTTGTTTACCGAGGTACAACAACTTACAAATTAAATGTTG GTGATTATTTTGTGCTGACATCACATACAGTAATGCCATTAAGTGCACCT ACACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGCTTATACCCAAC ACTCAATATCTCAGATGAGTTTTCTAGCAATGTTGCAAATTATCAAAAGG TTGGTATGCAAAAGTATTCTACACTCCAGGGACCACCTGGTACTGGTAAG AGTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTGCTCGCATAGT GTATACAGCTTGCTCTCATGCCGCTGTTGATGCACTATGTGAGAAGGCAT TAAAATATTTGCCTATAGATAAATGTAGTAGAATTATACCTGCACGTGCT CGTGTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATTAGAACAGTA TGTCTTTTGTACTGTAAATGCATTGCCTGAGACGACAGCAGATATAGTTG TCTTTGATGAAATTTCAATGGCCACAAATTATGATTTGAGTGTTGTCAAT GCCAGATTACGTGCTAAGCACTATGTGTACATTGGCGACCCTGCTCAATT ACCTGCACCACGCACATTGCTAACTAAGGGCACACTAGAACCAGAATATT TCAATTCAGTGTGTAGACTTATGAAAACTATAGGTCCAGACATGTTCCTC GGAACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGTGAGTGCTTT GGTTTATGATAATAAGCTTAAAGCACATAAAGACAAATCAGCTCAATGCT TTAAAATGTTTTATAAGGGTGTTATCACGCATGATGTTTCATCTGCAATT AACAGGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACGTAACCCTGC TTGGAGAAAAGCTGTCTTTATTTCACCTTATAATTCACAGAATGCTGTAG CCTCAAAGATTTTGGGACTACCAACTCAAACTGTTGATTCATCACAGGGC TCAGAATATGACTATGTCATATTCACTCAAACCACTGAAACAGCTCACTC TTGTAATGTAAACAGATTTAATGTTGCTATTACCAGAGCAAAAGTAGGCA TACTTTGCATAATGTCTGATAGAGACCTTTATGACAAGTTGCAATTTACA AGTCTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAGCTGAAAATGT AACAGGACTCTTTAAAGATTGTAGTAAGGTAATCACTGGGTTACATCCTA CACAGGCACCTACACACCTCAGTGTTGACACTAAATTCAAAACTGAAGGT TTATGTGTTGACATACCTGGCATACCTAAGGACATGACCTATAGAAGACT CATCTCTATGATGGGTTTTAAAATGAATTATCAAGTTAATGGTTACCCTA ACATGTTTATCACCCGCGAAGAAGCTATAAGACATGTACGTGCATGGATT GGCTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCTGTTGGTACCAA TTTACCTTTACAGCTAGGTTTTTCTACAGGTGTTAACCTAGTTGCTGTAC CTACAGGTTATGTTGATACACCTAATAATACAGATTTTTCCAGAGTTAGT GCTAAACCACCGCCTGGAGATCAATTTAAACACCTCATACCACTTATGTA CAAAGGACTTCCTTGGAATGTAGTGCGTATAAAGATTGTACAAATGTTAA GTGACACACTTAAAAATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCA CATGGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAATAGGACCTGA GCGCACCTGTTGTCTATGTGATAGACGTGCCACATGCTTTTCCACTGCTT CAGACACTTATGCCTGTTGGCATCATTCTATTGGATTTGATTACGTCTAT AATCCGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGTAACCTACA AAGCAACCATGATCTGTATTGTCAAGTCCATGGTAATGCACATGTAGCTA GTTGTGATGCAATCATGACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTT AAGCGTGTTGACTGGACTATTGAATATCCTATAATTGGTGATGAACTGAA GATTAATGCGGCTTGTAGAAAGGTTCAACACATGGTTGTTAAAGCTGCAT TATTAGCAGACAAATTCCCAGTTCTTCACGACATTGGTAACCCTAAAGCT ATTAAGTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTATGATGCACA GCCTTGTAGTGACAAAGCTTATAAAATAGAAGAATTATTCTATTCTTATG CCACACATTCTGACAAATTCACAGATGGTGTATGCCTATTTTGGAATTGC AATGTCGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTGACACTAG AGTGCTATCTAACCTTAACTTGCCTGGTTGTGATGGTGGCAGTTTGTATG TAAATAAACATGCATTCCACACACCAGCTTTTGATAAAAGTGCTTTTGTT AATTTAAAACAATTACCATTTTTCTATTACTCTGACAGTCCATGTGAGTC TCATGGAAAACAAGTAGTGTCAGATATAGATTATGTACCACTAAAGTCTG CTACGTGTATAACACGTTGCAATTTAGGTGGTGCTGTCTGTAGACATCAT GCTAATGAGTACAGATTGTATCTCGATGCTTATAACATGATGATCTCAGC TGGCTTTAGCTTGTGGGTTTACAAACAATTTGATACTTATAACCTCTGGA ACACTTTTACAAGACTTCAGAGTTTAGAAAATGTGGCTTTTAATGTTGTA AATAAGGGACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCTATCAT TAATAACACTGTTTACACAAAAGTTGATGGTGTTGATGTAGAATTGTTTG AAAATAAAACAACATTACCTGTTAATGTAGCATTTGAGCTTTGGGCTAAG CGCAACATTAAACCAGTACCAGAGGTGAAAATACTCAATAATTTGGGTGT GGACATTGCTGCTAATACTGTGATCTGGGACTACAAAAGAGATGCTCCAG CACATATATCTACTATTGGTGTTTGTTCTATGACTGACATAGCCAAGAAA CCAACTGAAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGTAGAGT TGATGGTCAAGTAGACTTATTTAGAAATGCCCGTAATGGTGTTCTTATTA CAGAAGGTAGTGTTAAAGGTTTACAACCATCTGTAGGTCCCAAACAAGCT AGTCTTAATGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAGTTCAA TTATTATAAGAAAGTTGATGGTGTTGTCCAACAATTACCTGAAACTTACT TTACTCAGAGTAGAAATTTACAAGAATTTAAACCCAGGAGTCAAATGGAA ATTGATTTCTTAGAATTAGCTATGGATGAATTCATTGAACGGTATAAATT AGAAGGCTATGCCTTCGAACATATCGTTTATGGAGATTTTAGTCATAGTC AGTTAGGTGGTTTACATCTACTGATTGGACTAGCTAAACGTTTTAAGGAA TCACCTTTTGAATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAAAA CTATTTCATAACAGATGCGCAAACAGGTTCATCTAAGTGTGTGTGTTCTG TTATTGATTTATTACTTGATGATTTTGTTGAAATAATAAAATCCCAAGAT TTATCTGTAGTTTCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAAT TTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAAACATTTTACCCAA AATTACAATCTAGTCAAGCGTGGCAACCGGGTGTTGCTATGCCTAATCTT TACAAAATGCAAAGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATGG TGATAGTGCAACATTACCTAAAGGCATAATGATGAATGTCGCAAAATATA CTCAACTGTGTCAATATTTAAACACATTAACATTAGCTGTACCCTATAAT ATGAGAGTTATACATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGG TACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGCTGCTTGTCGATT CAGATCTTAATGACTTTGTCTCTGATGCAGATTCAACTTTGATTGGTGAT TGTGCAACTGTACATACAGCTAATAAATGGGATCTCATTATTAGTGATAT GTACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGACTCTAAAGAGG GTTTTTTCACTTACATTTGTGGGTTTATACAACAAAAGCTAGCTCTTGGA GGTTCCGTGGCTATAAAGATAACAGAACATTCTTGGAATGCTGATCTTTA TAAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTGTTACTAATGTGA ATGCGTCATCATCTGAAGCATTTTTAATTGGATGTAATTATCTTGGCAAA CCACGCGAACAAATAGATGGTTATGTCATGCATGCAAATTACATATTTTG GAGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTTATTTGACATGA GTAAATTTCCCCTTAAATTAAGGGGTACTGCTGTTATGTCTTTAAAAGAA GGTCAAATCAATGATATGATTTTATCTCTTCTTAGTAAAGGTAGACTTAT AATTAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTCTTGTTAACA ACTAAACGAACAATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAG TCAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCCTGCATACACTA ATTCTTTCACACGTGGTGTTTATTACCCTGACAAAGTTTTCAGATCCTCA GTTTTACATTCAACTCAGGACTTGTTCTTACCTTTCTTTTCCAATGTTAC TTGGTTCCATGCTATACATGTCTCTGGGACCAATGGTACTAAGAGGTTTG ATAACCCTGTCCTACCATTTAATGATGGTGTTTATTTTGCTTCCACTGAG AAGTCTAACATAATAAGAGGCTGGATTTTTGGTACTACTTTAGATTCGAA GACCCAGTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTATTAAAG TCTGTGAATTTCAATTTTGTAATGATCCATTTTTGGGTGTTTATTACCAC AAAAACAACAAAAGTTGGATGGAAAGTGAGTTCAGAGTTTATTCTAGTGC GAATAATTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGACCTTG AAGGAAAACAGGGTAATTTCAAAAATCTTAGGGAATTTGTGTTTAAGAAT ATTGATGGTTATTTTAAAATATATTCTAAGCACACGCCTATTAATTTAGT GCGTGATCTCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGATTTGC CAATAGGTATTAACATCACTAGGTTTCAAACTTTACTTGCTTTACATAGA AGTTATTTGACTCCTGGTGATTCTTCTTCAGGTTGGACAGCTGGTGCTGC AGCTTATTATGTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATATA ATGAAAATGGAACCATTACAGATGCTGTAGACTGTGCACTTGACCCTCTC TCAGAAACAAAGTGTACGTTGAAATCCTTCACTGTAGAAAAAGGAATCTA TCAAACTTCTAACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATTTC CTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGA TTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGC TGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTT ATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTAT GCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGG GCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTA CAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGT GGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACC TTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTT GTAATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGT TTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACT TTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGT CTACTAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCAATGGTTTA ACAGGCACAGGTGTTCTTACTGAGTCTAACAAAAAGTTTCTGCCTTTCCA ACAATTTGGCAGAGACATTGCTGACACTACTGATGCTGTCCGTGATCCAC AGACACTTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGTGTCAGT GTTATAACACCAGGAACAAATACTTCTAACCAGGTTGCTGTTCTTTATCA GGATGTTAACTGCACAGAAGTCCCTGTTGCTATTCATGCAGATCAACTTA CTCCTACTTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAACACGT GCAGGCTGTTTAATAGGGGCTGAACATGTCAACAACTCATATGAGTGTGA CATACCCATTGGTGCAGGTATATGCGCTAGTTATCAGACTCAGACTAATT CTCCTCGGCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCTACACT ATGTCACTTGGTGCAGAAAATTCAGTTGCTTACTCTAATAACTCTATTGC CATACCCACAAATTTTACTATTAGTGTTACCACAGAAATTCTACCAGTGT CTATGACCAAGACATCAGTAGATTGTACAATGTACATTTGTGGTGATTCA ACTGAATGCAGCAATCTTTTGTTGCAATATGGCAGTTTTTGTACACAATT AAACCGTGCTTTAACTGGAATAGCTGTTGAACAAGACAAAAACACCCAAG AAGTTTTTGCACAAGTCAAACAAATTTACAAAACACCACCAATTAAAGAT TTTGGTGGTTTTAATTTTTCACAAATATTACCAGATCCATCAAAACCAAG CAAGAGGTCATTTATTGAAGATCTACTTTTCAACAAAGTGACACTTGCAG ATGCTGGCTTCATCAAACAATATGGTGATTGCCTTGGTGATATTGCTGCT AGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTTACTGTTTTGCCACC TTTGCTCACAGATGAAATGATTGCTCAATACACTTCTGCACTGTTAGCGG GTACAATCACTTCTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATA CCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTATTGGAGTTACACA GAATGTTCTCTATGAGAACCAAAAATTGATTGCCAACCAATTTAATAGTG CTATTGGCAAAATTCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGA AAACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTTAAACACGCTTGT TAAACAACTTAGCTCCAATTTTGGTGCAATTTCAAGTGTTTTAAATGATA TCCTTTCACGTCTTGACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTG ATCACAGGCAGACTTCAAAGTTTGCAGACATATGTGACTCAACAATTAAT TAGAGCTGCAGAAATCAGAGCTTCTGCTAATCTTGCTGCTACTAAAATGT CAGAGTGTGTACTTGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGC TATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGGTGTAGTCTTCTT GCATGTGACTTATGTCCCTGCACAAGAAAAGAACTTCACAACTGCTCCTG CCATTTGTCATGATGGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTT TCAAATGGCACACACTGGTTTGTAACACAAAGGAATTTTTATGAACCACA AATCATTACTACAGACAACACATTTGTGTCTGGTAACTGTGATGTTGTAA TAGGAATTGTCAACAACACAGTTTATGATCCTTTGCAACCTGAATTAGAC TCATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCATACATCACCAGA TGTTGATTTAGGTGACATCTCTGGCATTAATGCTTCAGTTGTAAACATTC AAAAAGAAATTGACCGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCT CTCATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTATATAAAATGGCC ATGGTACATTTGGCTAGGTTTTATAGCTGGCTTGATTGCCATAGTAATGG TGACAATTATGCTTTGCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGC TGTTGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGACTCTGAGCC AGTGCTCAAAGGAGTCAAATTACATTACACATAAACGAACTTATGGATTT GTTTATGAGAATCTTCACAATTGGAACTGTAACTTTGAAGCAAGGTGAAA TCAAGGATGCTACTCCTTCAGATTTTGTTCGCGCTACTGCAACGATACCG ATACAAGCCTCACTCCCTTTCGGATGGCTTATTGTTGGCGTTGCACTTCT TGCTGTTTTTCAGAGCGCTTCCAAAATCATAACCCTCAAAAAGAGATGGC AACTAGCACTCTCCAAGGGTGTTCACTTTGTTTGCAACTTGCTGTTGTTG TTTGTAACAGTTTACTCACACCTTTTGCTCGTTGCTGCTGGCCTTGAAGC CCCTTTTCTCTATCTTTATGCTTTAGTCTACTTCTTGCAGAGTATAAACT TTGTAAGAATAATAATGAGGCTTTGGCTTTGCTGGAAATGCCGTTCCAAA AACCCATTACTTTATGATGCCAACTATTTTCTTTGCTGGCATACTAATTG TTACGACTATTGTATACCTTACAATAGTGTAACTTCTTCAATTGTCATTA CTTCAGGTGATGGCACAACAAGTCCTATTTCTGAACATGACTACCAGATT GGTGGTTATACTGAAAAATGGGAATCTGGAGTAAAAGACTGTGTTGTATT ACACAGTTACTTCACTTCAGACTATTACCAGCTGTACTCAACTCAATTGA GTACAGACACTGGTGTTGAACATGTTACCTTCTTCATCTACAATAAAATT GTTGATGAGCCTGAAGAACATGTCCAAATTCACACAATCGACGGTTCATC CGGAGTTGTTAATCCAGTAATGGAACCAATTTATGATGAACCGACGACGA CTACTAGCGTGCCTTTGTAAGCACAAGCTGATGAGTACGAACTTATGTAC TCATTCGTTTCGGAAGAGACAGGTACGTTAATAGTTAATAGCGTACTTCT TTTTCTTGCTTTCGTGGTATTCTTGCTAGTTACACTAGCCATCCTTACTG CGCTTCGATTGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTTGTA AAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAATCTGAATTCTTCTAG AGTTCCTGATCTTCTGGTCTAAACGAACTAAATATTATATTAGTTTTTCT GTTTGGAACTTTAATTTTAGCCATGGCAGATTCCAACGGTACTATTACCG TTGAAGAGCTTAAAAAGCTCCTTGAACAATGGAACCTAGTAATAGGTTTC CTATTCCTTACATGGATTTGTCTTCTACAATTTGCCTATGCCAACAGGAA TAGGTTTTTGTATATAATTAAGTTAATTTTCCTCTGGCTGTTATGGCCAG TAACTTTAGCTTGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGATC ACCGGTGGAATTGCTATCGCAATGGCTTGTCTTGTAGGCTTGATGTGGCT CAGCTACTTCATTGCTTCTTTCAGACTGTTTGCGCGTACGCGTTCCATGT GGTCATTCAATCCAGAAACTAACATTCTTCTCAACGTGCCACTCCATGGC ACTATTCTGACCAGACCGCTTCTAGAAAGTGAACTCGTAATCGGAGCTGT GATCCTTCGTGGACATCTTCGTATTGCTGGACACCATCTAGGACGCTGTG ACATCAAGGACCTGCCTAAAGAAATCACTGTTGCTACATCACGAACGCTT TCTTATTACAAATTGGGAGCTTCGCAGCGTGTAGCAGGTGACTCAGGTTT TGCTGCATACAGTCGCTACAGGATTGGCAACTATAAATTAAACACAGACC ATTCCAGTAGCAGTGACAATATTGCTTTGCTTGTACAGTAAGTGACAACA GATGTTTCATCTCGTTGACTTTCAGGTTACTATAGCAGAGATATTACTAA TTATTATGAGGACTTTTAAAGTTTCCATTTGGAATCTTGATTACATCATA AACCTCATAATTAAAAATTTATCTAAGTCACTAACTGAGAATAAATATTC TCAATTAGATGAAGAGCAACCAATGGAGATTGATTAAACGAACATGAAAA TTATTCTTTTCTTGGCACTGATAACACTCGCTACTTGTGAGCTTTATCAC TACCAAGAGTGTGTTAGAGGTACAACAGTACTTTTAAAAGAACCTTGCTC TTCTGGAACATACGAGGGCAATTCACCATTTCATCCTCTAGCTGATAACA AATTTGCACTGACTTGCTTTAGCACTCAATTTGCTTTTGCTTGTCCTGAC GGCGTAAAACACGTCTATCAGTTACGTGCCAGATCAGTTTCACCTAAACT GTTCATCAGACAAGAGGAAGTTCAAGAACTTTACTCTCCAATTTTTCTTA TTGTTGCGGCAATAGTGTTTATAACACTTTGCTTCACACTCAAAAGAAAG ACAGAATGATTGAACTTTCATTAATTGACTTCTATTTGTGCTTTTTAGCC TTTCTGCTATTCCTTGTTTTAATTATGCTTATTATCTTTTGGTTCTCACT TGAACTGCAAGATCATAATGAAACTTGTCACGCCTAAACGAACATGAAAT TTCTTGTTTTCTTAGGAATCATCACAACTGTAGCTGCATTTCACCAAGAA TGTAGTTTACAGTCATGTACTCAACATCAACCATATGTAGTTGATGACCC GTGTCCTATTCACTTCTATTCTAAATGGTATATTAGAGTAGGAGCTAGAA AATCAGCACCTTTAATTGAATTGTGCGTGGATGAGGCTGGTTCTAAATCA CCCATTCAGTACATCGATATCGGTAATTATACAGTTTCCTGTTTACCTTT TACAATTAATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGTTGTT CGTTCTATGAAGACTTTTTAGAGTATCATGACGTTCGTGTTGTTTTAGAT TTCATCTAAACGAACAAACTAAAATGTCTGATAATGGACCCCAAAATCAG CGAAATGCACCCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGCAG TAACCAGAATGGAGAACGCAGTGGGGCGCGATCAAAACAACGTCGGCCCC AAGGTTTACCCAATAATACTGCGTCTTGGTTCACCGCTCTCACTCAACAT GGCAAGGAAGACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTAACAC CAATAGCAGTCCAGATGACCAAATTGGCTACTACCGAAGAGCTACCAGAC GAATTCGTGGTGGTGACGGTAAAATGAAAGATCTCAGTCCAAGATGGTAT TTCTACTACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGGTGCTAA CAAAGACGGCATCATATGGGTTGCAACTGAGGGAGCCTTGAATACACCAA AAGATCACATTGGCACCCGCAATCCTGCTAACAATGCTGCAATCGTGCTA CAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAGAAGGGAG CAGAGGCGGCAGTCAAGCCTCTTCTCGTTCCTCATCACGTAGTCGCAACA GTTCAAGAAATTCAACTCCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGA ATGGCTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTGCTTGACAG ATTGAACCAGCTTGAGAGCAAAATGTCTGGTAAAGGCCAACAACAACAAG GCCAAACTGTCACTAAGAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGG CAAAAACGTACTGCCACTAAAGCATACAATGTAACACAAGCTTTCGGCAG ACGTGGTCCAGAACAAACCCAAGGAAATTTTGGGGACCAGGAACTAATCA GACAAGGAACTGATTACAAACATTGGCCGCAAATTGCACAATTTGCCCCC AGCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATGGAAGTCACACC TTCGGGAACGTGGTTGACCTACACAGGTGCCATCAAATTGGATGACAAAG ATCCAAATTTCAAAGATCAAGTCATTTTGCTGAATAAGCATATTGACGCA TACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAAAAAGAAGAAGGC TGATGAAACTCAAGCCTTACCGCAGAGACAGAAGAAACAGCAAACTGTGA CTCTTCTTCCTGCTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAA TCCATGAGCAGTGCTGACTCAACTCAGGCCTAAACTCATGCAGACCACAC AAGGCAGATGGGCTATATAAACGTTTTCGCTTTTCCGTTTACGATATATA GTCTACTCTTGTGCAGAATGAATTCTCGTAACTACATAGCACAAGTAGAT GTAGTTAACTTTAATCTCACATAGCAATCTTTAATCAGTGTGTAACATTA GGGAGGACTTGAAAGAGCCACCACATTTTCACCGAGGCCACGCGGAGTAC GATCGAGTGTACAGTGAACAATGCTAGGGAGAGCTGCCTATATGGAAGAG CCCTAATGTGTAAAATTAATTTTAGTAGTGCTATCCCCATGTGATTTTAA TAGCTTCTTAGGAGAATGACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAA (SEQ ID NO: 1)

TABLE 5 SARS-CoV2 gene sequences Gene Start End Sequence orf1a   266 13483 ATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAACAC (SEQ ID ACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGAC NO: 2) GTGCTCGTACGTGGCTTTGGAGACTCCGTGGAGGAGG TCTTATCAGAGGCACGTCAACATCTTAAAGATGGCAC TTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTC AACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGAT GCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCT GGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGT GGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGGCGA AATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGA ACGGTAATAAAGGAGCTGGTGGCCATAGTTACGGCGC CGATCTAAAGTCATTTGACTTAGGCGACGAGCTTGGC ACTGATCCTTATGAAGATTTTCAAGAAAACTGGAACA CTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCG TGAGCTTAACGGAGGGGCATACACTCGCTATGTCGAT AACAACTTCTGTGGCCCTGATGGCTACCCTCTTGAGTG CATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTCAT GCACTTTGTCCGAACAACTGGACTTTATTGACACTAAG AGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAA TTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGA ATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAA TTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATT TCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGG TTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAAT TCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCA ACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCAT TGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTA AAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACT AAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAA ATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAAT TCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACC ATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGT GGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTA TGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCAC GTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGT TGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTT CTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATA TTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATT ATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGT GGAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAAC AAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAA GGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAAC AGAAATCAATACTGAGTCCTCTTTATGCATTTGCATCA GAGGCTGCTCGTGTTGTACGATCAATTTTCTCCCGCAC TCTTGAAACTGCTCAAAATTCTGTGCGTGTTTTACAGA AGGCCGCTATAACAATACTAGATGGAATTTCACAGTA TTCACTGAGACTCATTGATGCTATGATGTTCACATCTG ATTTGGCTACTAACAATCTAGTTGTAATGGCCTACATT ACAGGTGGTGTTGTTCAGTTGACTTCGCAGTGGCTAAC TAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCG TCCTTGATTGGCTTGAAGAGAAGTTTAAGGAAGGTGT AGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTA TCTCAACCTGTGCTTGTGAAATTGTCGGTGGACAAATT GTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGA CATTCTTTAAGCTTGTAAATAAATTTTTGGCTTTGTGT GCTGACTCTATCATTATTGGTGGAGCTAAACTTAAAGC CTTGAATTTAGGTGAAACATTTGTCACGCACTCAAAG GGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAA CTGGCCTACTCATGCCTCTAAAAGCCCCAAAAGAAAT TATCTTCTTAGAGGGAGAAACACTTCCCACAGAAGTG TTAACAGAGGAAGTTGTCTTGAAAACTGGTGATTTAC AACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGC TCCATTGGTTGGTACACCAGTTTGTATTAACGGGCTTA TGTTGCTCGAAATCAAAGACACAGAAAAGTACTGTGC CCTTGCACCTAATATGATGGTAACAAACAATACCTTCA CACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGG TGATGACACTGTGATAGAAGTGCAAGGTTACAAGAGT GTGAATATCACTTTTGAACTTGATGAAAGGATTGATA AAGTACTTAATGAGAAGTGCTCTGCCTATACAGTTGA ACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTG GCAGATGCTGTCATAAAAACTTTGCAACCAGTATCTG AATTACTTACACCACTGGGCATTGATTTAGATGAGTGG AGTATGGCTACATACTACTTATTTGATGAGTCTGGTGA GTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACC CTCCAGATGAGGATGAAGAAGAAGGTGATTGTGAAGA AGAAGAGTTTGAGCCATCAACTCAATATGAGTATGGT ACTGAAGATGATTACCAAGGTAAACCTTTGGAATTTG GTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCA AGAAGAAGATTGGTTAGATGATGATAGTCAACAAACT GTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAA CTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTA GAGATGGAACTTACACCAGTTGTTCAGACTATTGAAG TGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAAT GTATACATTAAAAATGCAGACATTGTGGAAGAAGCTA AAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAA TGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCC TTAAATAAGGCTACTAACAATGCCATGCAAGTTGAAT CTGATGATTACATAGCTACTAATGGACCACTTAAAGT GGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTA AACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAA GGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAA ATTTTAATCAGCACGAAGTTCTACTTGCACCATTATTA TCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTT AAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACT TAGCTGTCTTTGATAAAAATCTCTATGACAAACTTGTT TCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTG AACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAA GCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAG AGAAAACAAGATGATAAGAAAATCAAAGCTTGTGTTG AAGAAGTTACAACAACTCTGGAAGAAACTAAGTTCCT CACAGAAAACTTGTTACTTTATATTGACATTAATGGCA ATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATT GACATCACTTTCTTAAAGAAAGATGCTCCATATATAGT GGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGTGG TTATACCTACTAAAAAGGCTGGTGGCACTACTGAAAT GCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAAT TATATAACCACTTACCCGGGTCAGGGTTTAAATGGTTA CACTGTAGAGGAGGCAAAGACAGTGCTTAAAAAGTGT AAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAA TGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATT TGCGAGAAATGCTTGCACATGCAGAAGAAACACGCAA ATTAATGCCTGTCTGTGTGGAAACTAAAGCCATAGTTT CAACTATACAGCGTAAATATAAGGGTATTAAAATACA AGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTT ACACCAGTAAAACAACTGTAGCGTCACTTATCAACAC ACTTAACGATCTAAATGAAACTCTTGTTACAATGCCAC TTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGC TGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACA GTTTCTGTTTCTTCACCTGATGCTGTTACAGCGTATAA TGGTTATCTTACTTCTTCTTCTAAAACACCTGAAGAAC ATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTAT AGAATTTCTTAAGAGAGGTGATAAAAGTGTATATTAC ACTAGTAATCCTACCACATTCCACCTAGATGGTGAAGT TATCACCTTTGACAATCTTAAGACACTTCTTTCTTTGA GAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGA CAACATTAACCTCCACACGCAAGTTGTGGACATGTCA ATGACATATGGACAACAGTTTGGTCCAACTTATTTGGA TGGAGCTGATGTTACTAAAATAAAACCTCATAATTCA CATGAAGGTAAAACATTTTATGTTTTACCTAATGATGA CACTCTACGTGTTGAGGCTTTTGAGTACTACCACACAA CTGATCCTAGTTTTCTGGGTAGGTACATGTCAGCATTA AATCACACTAAAAAGTGGAAATACCCACAAGTTAATG GTTTAACTTCTATTAAATGGGCAGATAACAACTGTTAT CTTGCCACTGCATTGTTAACACTCCAACAAATAGAGTT GAAGTTTAATCCACCTGCTCTACAAGATGCTTATTACA GAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGCACT TATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTA GGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAAC ATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTG GTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTA AGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCT TATGAACAATTTAAGAAAGGTGTTCAGATACCTTGTA CGTGTGGTAAACAAGCTACAAAATATCTAGTACAACA GGAGTCACCTTTTGTTATGATGTCAGCACCACCTGCTC AGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGT GAGTACACTGGTAATTACCAGTGTGGTCACTATAAAC ATATAACTTCTAAAGAAACTTTGTATTGCATAGACGGT GCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTAT TACGGATGTTTTCTACAAAGAAAACAGTTACACAACA ACCATAAAACCAGTTACTTATAAATTGGATGGTGTTGT TTGTACAGAAATTGACCCTAAGTTGGACAATTATTATA AGAAAGACAATTCTTATTTCACAGAGCAACCAATTGA TCTTGTACCAAACCAACCATATCCAAACGCAAGCTTC GATAATTTTAAGTTTGTATGTGATAATATCAAATTTGC TGATGATTTAAACCAGTTAACTGGTTATAAGAAACCT GCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGACTT AAATGGTGATGTGGTGGCTATTGATTATAAACACTAC ACACCCTCTTTTAAGAAAGGAGCTAAATTGTTACATA AACCTATTGTTTGGCATGTTAACAATGCAACTAATAAA GCCACGTATAAACCAAATACCTGGTGTATACGTTGTCT TTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTT GATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATA ATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGA AGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTT GAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACA TTATACTTAAACCAGCAAATAATAGTTTAAAAATTAC AGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTAT GTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGA ATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTC ATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACT ATAGCTAATTATGCTAAGCCTTTTCTTAACAAAGTTGT TAGTACAACTACTAACATAGTTACACGGTGTTTAAACC GTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTA TTGCTACAATTGTGTACTTTTACTAGAAGTACAAATTC TAGAATTAAAGCATCTATGCCGACTACTATAGCAAAGA ATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGAGGCT TCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACT GATAAATATTATAATTTGGTTTTTACTATTAAGTGTTT GCCTAGGTTCTTTAATCTACTCAACCGCTGCTTTAGGT GTTTTAATGTCTAATTTAGGCATGCCTTCTTACTGTAC TGGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCA CTATTGCAACCTACTGTACTGGTTCTATACCTTGTAGT GTTTGTCTTAGTGGTTTAGATTCTTTAGACACCTATCC TTCTTTAGAAACTATACAAATTACCATTTCATCTTTTA AATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGG TTTTTGGCATATATTCTTTTCACTAGGTTTTTCTATGT ACTTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCT ATTTTGCAGTACATTTTATTAGTAATTCTTGGCTTAT GTGGTTAATAATTAATCTTGTACAAATGGCCCCGATTT CAGCTATGGTTAGAATGTACATCTTCTTTGCATCATT TTATTATGTATGGAAAAGTTATGTGCATGTTGTAGACG GTTGTAATTCATCAACTTGTATGATGTGTTACAAACGT AATAGAGCAACAAGAGTCGAATGTACAACTATTGTTAA TGGTGTTAGAAGGTCCTTTTATGTCTATGCTAATGGAG GTAAAGGCTTTTGCAAACTACACAATTGGAATTGTGTT AATTGTGATACATTCTGTGCTGGTAGTACATTTATTAG TGATGAAGTTGCGAGAGACTTGTCACTACAGTTTAAA AGACCAATAAATCCTACTGACCAGTCTTCTTACATCGT TGATAGTGTTACAGTGAAGAATGGTTCCATCCATCTTT ACTTTGATAAAGCTGGTCAAAAGACTTATGAAAGACATTC TCTCTCTCATTTTGTTAACTTAGACAACCTGAGAGCTAA TAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTT TTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAA ATCAGCGTCTGTTTACTACAGTCAGCTTATGTGTCAAC CTATACTGTTACTAGATCAGGCATTAGTGTCTGATGTT GGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATG CTTACGTTAATACGTTTTCATCAACTTTTAACGTACCA ATGGAAAAACTCAAAACACTAGTTGCAACTGCAGAAG CTGAACTTGCAAAGAATGTGTCCTTAGACAATGTCTTA TCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGA TTCAGATGTAGAAACTAAAGATGTTGTTGAATGTCTTA AATTGTCACATCAATCTGACATAGAAGTTACTGGCGA TAGTTGTAATAACTATATGCTCACCTATAACAAAGTTG AAAACATGACACCCCGTGACCTTGGTGCTTGTATTGAC TGTAGTGCGCGTCATATTAATGCGCAGGTAGCAAAAA GTCACAACATTGCTTTGATATGGAACGTTAAAGATTTC ATGTCATTGTCTGAACAACTACGAAAACAAATACGTA GTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACA TGTGCAACTACTAGACAAGTTGTTAATGTTGTAACAAC AAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAAT TGGTTGAAGCAGTTAATTAAAGTTACACTTGTGTTCCT TTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCA TGTCATGTCTAAACATACTGACTTTTCAAGTGAAATCA TAGGATACAAGGCTATTGATGGTGGTGTCACTCGTGA CATAGCATCTACAGATACTTGTTTTGCTAACAAACATG CTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGT TATACTAATGACAAAGCTTGCCCATTGATTGCTGCAGT CATAACAAGAGAAGTGGGTTTTGTCGTGCCTGGTTTGC CTGGCACGATATTACGCACAACTAATGGTGACTTTTTG CATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACAT CTGTTACACACCATCAAAACTTATAGAGTACACTGACT TTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACA ATTTTTAAAGATGCTTCTGGTAAGCCAGTACCATATTG TTATGATACCAATGTACTAGAAGGTTCTGTTGCTTATG AAAGTTTACGCCCTGACACACGTTATGTGCTCATGGAT GGCTCTATTATTCAATTTCCTAACACCTACCTTGAAGG TTCTGTTAGAGTGGTAACAACTTTTGATTCTGAGTACT GTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGT TTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATG ATTATTACAGATCTTTACCAGGAGTTTTCTGTGGTGTA GATGCTGTAAATTTACTTACTAATATGTTTACACCACT AATTCAACCTATTGGTGCTTTGGACATATCAGCATCTA TAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGC CTTGCCTACTATTTTATGAGGTTTAGAAGAGCTTTTGG TGAATACAGTCATGTAGTTGCCTTTAATACTTTACTAT TCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTT ACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGT ACTTGACATTTTATCTTACTAATGATGTTTCTTTTTTAG CACATATTCAGTGGATGGTTATGTTCACACCTTTAGTA CCTTTCTGGATAACAATTGCTTATATCATTTGTATTTCC ACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAA GAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTT TTGAAGAAGCTGCGCTGTGCACCTTTTTGTTAAATAAA GAAATGTATCTAAAGTTGCGTAGTGATGTGCTATTACC TCTTACGCAATATAATAGATACTTAGCTCTTTATAATA AGTACAAGTATTTTAGTGGAGCAATGGATACAACTAG CTACAGAGAAGCTGCTTGTTGTCATCTCGCAAAGGCTC TCAATGACTTCAGTAACTCAGGTTCTGATGTTCTTTAC CAACCACCACAAACCTCTATCACCTCAGCTGTTTTGCA GAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAA GTTGAGGGTTGTATGGTACAAGTAACTTGTGGTACAA CTACACTTAACGGTCTTTGGCTTGATGACGTAGTTTAC TGTCCAAGACATGTGATCTGCACCTCTGAAGACATGCT TAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTA ATCATAATTTCTTGGTACAGGCTGGTAATGTTCAACTC AGGGTTATTGGACATTCTATGCAAAATTGTGTACTTAA GCTTAAGGTTGATACAGCCAATCCTAAGACACCTAAG TATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTC AGTGTTAGCTTGTTACAATGGTTCACCATCTGGTGTTT ACCAATGTGCTATGAGGCCCAATTTCACTATTAAGGGT TCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAA CATAGATTATGACTGTGTCTCTTTTTGTTACATGCACC ATATGGAATTACCAACTGGAGTTCATGCTGGCACAGA CTTAGAAGGTAACTTTTATGGACCTTTTGTTGACAGGC AAACAGCACAAGCAGCTGGTACGGACACAACTATTAC AGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAA ATGGAGACAGGTGGTTTCTCAATCGATTTACCACAACT CTTAATGACTTTAACCTTGTGGCTATGAAGTACAATTA TGAACCTCTAACACAAGACCATGTTGACATACTAGGA CCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATAT GTGTGCTTCATTAAAAGAATTACTGCAAAATGGTATG AATGGACGTACCATATTGGGTAGTGCTTTATTAGAAG ATGAATTTACACCTTTTGATGTTGTTAGACAATGCTCA GGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCA AGGGTACACACCACTGGTTGTTACTCACAATTTTGACT TCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTT GTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGC TATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGT TTGTCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGT TACCTTCTCTTGCCACTGTAGCTTATTTTAATATGGTCT ATATGCCTGCTAGTTGGGTGATGCGTATTATGACATGG TTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGCT AAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTAC TAATCCTTATGACAGCAAGAACTGTGTATGATGATGG TGCTAGGAGAGTGTGGACACTTATGAATGTCTTGACA CTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATCA AGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTT CTAACTACTCAGGTGTAGTTACAACTGTCATGTTTTTG GCCAGAGGTATTGTTTTTATGTGTGTTGAGTATTGCCC TATTTTCTTCATAACTGGTAATACACTTCAGTGTATAA TGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTT ACTTTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGAC TGACTCTTGGTGTTTATGATTACTTAGTTTCTACACAG GAGTTTAGATATATGAATTCACAGGGACTACTCCCAC CCAAGAATAGCATAGATGCCTTCAAACTCAACATTAA ATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGTAG CCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCAC ATCAGTAGTCTTACTCTCAGTTTTGCAACAACTCAGAG TAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAG TTACACAATGACATTCTCTTAGCTAAAGATACTACTGA AGCCTTTGAAAAAATGGTTTCACTACTTTCTGTTTTGC TTTCCATGCAGGGTGCTGTAGACATAAACAAGCTTTGT GAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTA TAGCCTCAGAGTTTAGTTCCCTTCCATCATATGCAGCT TTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGC TAATGGTGATTCTGAAGTTGTTCTTAAAAAGTTGAAGA AGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGAT GCAGCCATGCAACGTAAGTTGGAAAAGATGGCTGATC AAGCTATGACCCAAATGTATAAACAGGCTAGATCTGA GGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACA ATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGC ACTCAACAACATTATCAACAATGCAAGAGATGGTTGT GTTCCCTTGAACATAATACCTCTTACAACAGCAGCCAA ACTAATGGTTGTCATACCAGACTATAACACATATAAA AATACGTGTGATGGTACAACATTTACTTATGCATCAGC ATTGTGGGAAATCCAACAGGTTGTAGATGCAGATAGT AAAATTGTTCAACTTAGTGAAATTAGTATGGACAATTC ACCTAATTTAGCATGGCCTCTTATTGTAACAGCTTTAA GGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCT TAGTCCTGTTGCACTACGACAGATGTCTTGTGCTGCCG GTACTACACAAACTGCTTGCACTGATGACAATGCGTT AGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTA CTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGC TAGATTCCCTAAGAGTGATGGAACTGGTACTATCTATA CAGAACTGGAACCACCTTGTAGGTTTGTTACAGACAC ACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTATTA AAGGATTAAACAACCTAAATAGAGGTATGGTACTTGG TAGTTTAGCTGCCACAGTACGTCTACAAGCTGGTAATG CAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTC TGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGA TTATCTAGCTAGTGGGGGACAACCAATCACTAATTGT GTTAAGATGTTGTGTACACACACTGGTACTGGTCAGG CAATAACAGTTACACCGGAAGCCAATATGGATCAAGA ATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTT GCCACATAGATCATCCAAATCCTAAAGGATTTTGTGA CTTAAAAGGTAAGTATGTACAAATACCTACAACTTGT GCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGT CTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTA GTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCT GATGCACAATCGTTTTTAAACGGGTTTGCGGTGTA orf1ab  266 21555 ATGGAGAGCCTTGTCCCTGGTTTCAACGAGAAAACAC (SEQ ID ACGTCCAACTCAGTTTGCCTGTTTTACAGGTTCGCGAC NO: 3) GTGCTCGTACGTGGCTTTGGAGACTCCGTGGAGGAGG TCTTATCAGAGGCACGTCAACATCTTAAAGATGGCAC TTGTGGCTTAGTAGAAGTTGAAAAAGGCGTTTTGCCTC AACTTGAACAGCCCTATGTGTTCATCAAACGTTCGGAT GCTCGAACTGCACCTCATGGTCATGTTATGGTTGAGCT GGTAGCAGAACTCGAAGGCATTCAGTACGGTCGTAGT GGTGAGACACTTGGTGTCCTTGTCCCTCATGTGGGCGA AATACCAGTGGCTTACCGCAAGGTTCTTCTTCGTAAGA ACGGTAATAAAGGAGCTGGTGGCCATAGTTACGGCGC CGATCTAAAGTCATTTGACTTAGGCGACGAGCTTGGC ACTGATCCTTATGAAGATTTTCAAGAAAACTGGAACA CTAAACATAGCAGTGGTGTTACCCGTGAACTCATGCG TGAGCTTAACGGAGGGGCATACACTCGCTATGTCGAT AACAACTTCTGTGGCCCTGATGGCTACCCTCTTGAGTG CATTAAAGACCTTCTAGCACGTGCTGGTAAAGCTTCAT GCACTTTGTCCGAACAACTGGACTTTATTGACACTAAG AGGGGTGTATACTGCTGCCGTGAACATGAGCATGAAA TTGCTTGGTACACGGAACGTTCTGAAAAGAGCTATGA ATTGCAGACACCTTTTGAAATTAAATTGGCAAAGAAA TTTGACACCTTCAATGGGGAATGTCCAAATTTTGTATT TCCCTTAAATTCCATAATCAAGACTATTCAACCAAGGG TTGAAAAGAAAAAGCTTGATGGCTTTATGGGTAGAAT TCGATCTGTCTATCCAGTTGCGTCACCAAATGAATGCA ACCAAATGTGCCTTTCAACTCTCATGAAGTGTGATCAT TGTGGTGAAACTTCATGGCAGACGGGCGATTTTGTTA AAGCCACTTGCGAATTTTGTGGCACTGAGAATTTGACT AAAGAAGGTGCCACTACTTGTGGTTACTTACCCCAAA ATGCTGTTGTTAAAATTTATTGTCCAGCATGTCACAAT TCAGAAGTAGGACCTGAGCATAGTCTTGCCGAATACC ATAATGAATCTGGCTTGAAAACCATTCTTCGTAAGGGT GGTCGCACTATTGCCTTTGGAGGCTGTGTGTTCTCTTA TGTTGGTTGCCATAACAAGTGTGCCTATTGGGTTCCAC GTGCTAGCGCTAACATAGGTTGTAACCATACAGGTGT TGTTGGAGAAGGTTCCGAAGGTCTTAATGACAACCTT CTTGAAATACTCCAAAAAGAGAAAGTCAACATCAATA TTGTTGGTGACTTTAAACTTAATGAAGAGATCGCCATT ATTTTGGCATCTTTTTCTGCTTCCACAAGTGCTTTTGTG GAAACTGTGAAAGGTTTGGATTATAAAGCATTCAAAC AAATTGTTGAATCCTGTGGTAATTTTAAAGTTACAAAA GGAAAAGCTAAAAAAGGTGCCTGGAATATTGGTGAAC AGAAATCAATACTGAGTCCTCTTTATGCATTTGCATCA GAGGCTGCTCGTGTTGTACGATCAATTTTCTCCCGCAC TCTTGAAACTGCTCAAAATTCTGTGCGTGTTTTACAGA AGGCCGCTATAACAATACTAGATGGAATTTCACAGTA TTCACTGAGACTCATTGATGCTATGATGTTCACATCTG ATTTGGCTACTAACAATCTAGTTGTAATGGCCTACATT ACAGGTGGTGTTGTTCAGTTGACTTCGCAGTGGCTAAC TAACATCTTTGGCACTGTTTATGAAAAACTCAAACCCG TCCTTGATTGGCTTGAAGAGAAGTTTAAGGAAGGTGT AGAGTTTCTTAGAGACGGTTGGGAAATTGTTAAATTTA TCTCAACCTGTGCTTGTGAAATTGTCGGTGGACAAATT GTCACCTGTGCAAAGGAAATTAAGGAGAGTGTTCAGA CATTCTTTAAGCTTGTAAATAAATTTTTGGCTTTGTGT GCTGACTCTATCATTATTGGTGGAGCTAAACTTAAAGC CTTGAATTTAGGTGAAACATTTGTCACGCACTCAAAG GGATTGTACAGAAAGTGTGTTAAATCCAGAGAAGAAA CTGGCCTACTCATGCCTCTAAAAGCCCCAAAAGAAAT TATCTTCTTAGAGGGAGAAACACTTCCCACAGAAGTG TTAACAGAGGAAGTTGTCTTGAAAACTGGTGATTTAC AACCATTAGAACAACCTACTAGTGAAGCTGTTGAAGC TCCATTGGTTGGTACACCAGTTTGTATTAACGGGCTTA TGTTGCTCGAAATCAAAGACACAGAAAAGTACTGTGC CCTTGCACCTAATATGATGGTAACAAACAATACCTTCA CACTCAAAGGCGGTGCACCAACAAAGGTTACTTTTGG TGATGACACTGTGATAGAAGTGCAAGGTTACAAGAGT GTGAATATCACTTTTGAACTTGATGAAAGGATTGATA AAGTACTTAATGAGAAGTGCTCTGCCTATACAGTTGA ACTCGGTACAGAAGTAAATGAGTTCGCCTGTGTTGTG GCAGATGCTGTCATAAAAACTTTGCAACCAGTATCTG AATTACTTACACCACTGGGCATTGATTTAGATGAGTGG AGTATGGCTACATACTACTTATTTGATGAGTCTGGTGA GTTTAAATTGGCTTCACATATGTATTGTTCTTTCTACCC TCCAGATGAGGATGAAGAAGAAGGTGATTGTGAAGA AGAAGAGTTTGAGCCATCAACTCAATATGAGTATGGT ACTGAAGATGATTACCAAGGTAAACCTTTGGAATTTG GTGCCACTTCTGCTGCTCTTCAACCTGAAGAAGAGCA AGAAGAAGATTGGTTAGATGATGATAGTCAACAAACT GTTGGTCAACAAGACGGCAGTGAGGACAATCAGACAA CTACTATTCAAACAATTGTTGAGGTTCAACCTCAATTA GAGATGGAACTTACACCAGTTGTTCAGACTATTGAAG TGAATAGTTTTAGTGGTTATTTAAAACTTACTGACAAT GTATACATTAAAAATGCAGACATTGTGGAAGAAGCTA AAAAGGTAAAACCAACAGTGGTTGTTAATGCAGCCAA TGTTTACCTTAAACATGGAGGAGGTGTTGCAGGAGCC TTAAATAAGGCTACTAACAATGCCATGCAAGTTGAAT CTGATGATTACATAGCTACTAATGGACCACTTAAAGT GGGTGGTAGTTGTGTTTTAAGCGGACACAATCTTGCTA AACACTGTCTTCATGTTGTCGGCCCAAATGTTAACAAA GGTGAAGACATTCAACTTCTTAAGAGTGCTTATGAAA ATTTTAATCAGCACGAAGTTCTACTTGCACCATTATTA TCAGCTGGTATTTTTGGTGCTGACCCTATACATTCTTT AAGAGTTTGTGTAGATACTGTTCGCACAAATGTCTACT TAGCTGTCTTTGATAAAAATCTCTATGACAAACTTGTT TCAAGCTTTTTGGAAATGAAGAGTGAAAAGCAAGTTG AACAAAAGATCGCTGAGATTCCTAAAGAGGAAGTTAA GCCATTTATAACTGAAAGTAAACCTTCAGTTGAACAG AGAAAACAAGATGATAAGAAAATCAAAGCTTGTGTTG AAGAAGTTACAACAACTCTGGAAGAAACTAAGTTCCT CACAGAAAACTTGTTACTTTATATTGACATTAATGGCA ATCTTCATCCAGATTCTGCCACTCTTGTTAGTGACATT GACATCACTTTCTTAAAGAAAGATGCTCCATATATAGT GGGTGATGTTGTTCAAGAGGGTGTTTTAACTGCTGTGG TTATACCTACTAAAAAGGCTGGTGGCACTACTGAAAT GCTAGCGAAAGCTTTGAGAAAAGTGCCAACAGACAAT TATATAACCACTTACCCGGGTCAGGGTTTAAATGGTTA CACTGTAGAGGAGGCAAAGACAGTGCTTAAAAAGTGT AAAAGTGCCTTTTACATTCTACCATCTATTATCTCTAA TGAGAAGCAAGAAATTCTTGGAACTGTTTCTTGGAATT TGCGAGAAATGCTTGCACATGCAGAAGAAACACGCAA ATTAATGCCTGTCTGTGTGGAAACTAAAGCCATAGTTT CAACTATACAGCGTAAATATAAGGGTATTAAAATACA AGAGGGTGTGGTTGATTATGGTGCTAGATTTTACTTTT ACACCAGTAAAACAACTGTAGCGTCACTTATCAACAC ACTTAACGATCTAAATGAAACTCTTGTTACAATGCCAC TTGGCTATGTAACACATGGCTTAAATTTGGAAGAAGC TGCTCGGTATATGAGATCTCTCAAAGTGCCAGCTACA GTTTCTGTTTCTTCACCTGATGCTGTTACAGCGTATAA TGGTTATCTTACTTCTTCTTCTAAAACACCTGAAGAAC ATTTTATTGAAACCATCTCACTTGCTGGTTCCTATAAA GATTGGTCCTATTCTGGACAATCTACACAACTAGGTAT AGAATTTCTTAAGAGAGGTGATAAAAGTGTATATTAC ACTAGTAATCCTACCACATTCCACCTAGATGGTGAAGT TATCACCTTTGACAATCTTAAGACACTTCTTTCTTTGA GAGAAGTGAGGACTATTAAGGTGTTTACAACAGTAGA CAACATTAACCTCCACACGCAAGTTGTGGACATGTCA ATGACATATGGACAACAGTTTGGTCCAACTTATTTGGA TGGAGCTGATGTTACTAAAATAAAACCTCATAATTCA CATGAAGGTAAAACATTTTATGTTTTACCTAATGATGA CACTCTACGTGTTGAGGCTTTTGAGTACTACCACACAA CTGATCCTAGTTTTCTGGGTAGGTACATGTCAGCATTA AATCACACTAAAAAGTGGAAATACCCACAAGTTAATG GTTTAACTTCTATTAAATGGGCAGATAACAACTGTTAT CTTGCCACTGCATTGTTAACACTCCAACAAATAGAGTT GAAGTTTAATCCACCTGCTCTACAAGATGCTTATTACA GAGCAAGGGCTGGTGAAGCTGCTAACTTTTGTGCACT TATCTTAGCCTACTGTAATAAGACAGTAGGTGAGTTA GGTGATGTTAGAGAAACAATGAGTTACTTGTTTCAAC ATGCCAATTTAGATTCTTGCAAAAGAGTCTTGAACGTG GTGTGTAAAACTTGTGGACAACAGCAGACAACCCTTA AGGGTGTAGAAGCTGTTATGTACATGGGCACACTTTCT TATGAACAATTTAAGAAAGGTGTTCAGATACCTTGTA CGTGTGGTAAACAAGCTACAAAATATCTAGTACAACA GGAGTCACCTTTTGTTATGATGTCAGCACCACCTGCTC AGTATGAACTTAAGCATGGTACATTTACTTGTGCTAGT GAGTACACTGGTAATTACCAGTGTGGTCACTATAAAC ATATAACTTCTAAAGAAACTTTGTATTGCATAGACGGT GCTTTACTTACAAAGTCCTCAGAATACAAAGGTCCTAT TACGGATGTTTTCTACAAAGAAAACAGTTACACAACA ACCATAAAACCAGTTACTTATAAATTGGATGGTGTTGT TTGTACAGAAATTGACCCTAAGTTGGACAATTATTATA AGAAAGACAATTCTTATTTCACAGAGCAACCAATTGA TCTTGTACCAAACCAACCATATCCAAACGCAAGCTTC GATAATTTTAAGTTTGTATGTGATAATATCAAATTTGC TGATGATTTAAACCAGTTAACTGGTTATAAGAAACCT GCTTCAAGAGAGCTTAAAGTTACATTTTTCCCTGACTT AAATGGTGATGTGGTGGCTATTGATTATAAACACTAC ACACCCTCTTTTAAGAAAGGAGCTAAATTGTTACATA AACCTATTGTTTGGCATGTTAACAATGCAACTAATAAA GCCACGTATAAACCAAATACCTGGTGTATACGTTGTCT TTGGAGCACAAAACCAGTTGAAACATCAAATTCGTTT GATGTACTGAAGTCAGAGGACGCGCAGGGAATGGATA ATCTTGCCTGCGAAGATCTAAAACCAGTCTCTGAAGA AGTAGTGGAAAATCCTACCATACAGAAAGACGTTCTT GAGTGTAATGTGAAAACTACCGAAGTTGTAGGAGACA TTATACTTAAACCAGCAAATAATAGTTTAAAAATTAC AGAAGAGGTTGGCCACACAGATCTAATGGCTGCTTAT GTAGACAATTCTAGTCTTACTATTAAGAAACCTAATGA ATTATCTAGAGTATTAGGTTTGAAAACCCTTGCTACTC ATGGTTTAGCTGCTGTTAATAGTGTCCCTTGGGATACT ATAGCTAATTATGCTAAGCCTTTTCTTAACAAAGTTGT TAGTACAACTACTAACATAGTTACACGGTGTTTAAACC GTGTTTGTACTAATTATATGCCTTATTTCTTTACTTTAT TGCTACAATTGTGTACTTTTACTAGAAGTACAAATTCT AGAATTAAAGCATCTATGCCGACTACTATAGCAAAGA ATACTGTTAAGAGTGTCGGTAAATTTTGTCTAGAGGCT TCATTTAATTATTTGAAGTCACCTAATTTTTCTAAACT GATAAATATTATAATTTGGTTTTTACTATTAAGTGTTT GCCTAGGTTCTTTAATCTACTCAACCGCTGCTTTAGGT GTTTTAATGTCTAATTTAGGCATGCCTTCTTACTGTACT GGTTACAGAGAAGGCTATTTGAACTCTACTAATGTCA CTATTGCAACCTACTGTACTGGTTCTATACCTTGTAGT GTTTGTCTTAGTGGTTTAGATTCTTTAGACACCTATCCT TCTTTAGAAACTATACAAATTACCATTTCATCTTTTAA ATGGGATTTAACTGCTTTTGGCTTAGTTGCAGAGTGGT TTTTGGCATATATTCTTTTCACTAGGTTTTTCTATGTAC TTGGATTGGCTGCAATCATGCAATTGTTTTTCAGCTAT TTTGCAGTACATTTTATTAGTAATTCTTGGCTTATGTG GTTAATAATTAATCTTGTACAAATGGCCCCGATTTCAG CTATGGTTAGAATGTACATCTTCTTTGCATCATTTTATT ATGTATGGAAAAGTTATGTGCATGTTGTAGACGGTTGT AATTCATCAACTTGTATGATGTGTTACAAACGTAATAG AGCAACAAGAGTCGAATGTACAACTATTGTTAATGGT GTTAGAAGGTCCTTTTATGTCTATGCTAATGGAGGTAA AGGCTTTTGCAAACTACACAATTGGAATTGTGTTAATT GTGATACATTCTGTGCTGGTAGTACATTTATTAGTGAT GAAGTTGCGAGAGACTTGTCACTACAGTTTAAAAGAC CAATAAATCCTACTGACCAGTCTTCTTACATCGTTGAT AGTGTTACAGTGAAGAATGGTTCCATCCATCTTTACTT TGATAAAGCTGGTCAAAAGACTTATGAAAGACATTCT CTCTCTCATTTTGTTAACTTAGACAACCTGAGAGCTAA TAACACTAAAGGTTCATTGCCTATTAATGTTATAGTTT TTGATGGTAAATCAAAATGTGAAGAATCATCTGCAAA ATCAGCGTCTGTTTACTACAGTCAGCTTATGTGTCAAC CTATACTGTTACTAGATCAGGCATTAGTGTCTGATGTT GGTGATAGTGCGGAAGTTGCAGTTAAAATGTTTGATG CTTACGTTAATACGTTTTCATCAACTTTTAACGTACCA ATGGAAAAACTCAAAACACTAGTTGCAACTGCAGAAG CTGAACTTGCAAAGAATGTGTCCTTAGACAATGTCTTA TCTACTTTTATTTCAGCAGCTCGGCAAGGGTTTGTTGA TTCAGATGTAGAAACTAAAGATGTTGTTGAATGTCTTA AATTGTCACATCAATCTGACATAGAAGTTACTGGCGA TAGTTGTAATAACTATATGCTCACCTATAACAAAGTTG AAAACATGACACCCCGTGACCTTGGTGCTTGTATTGAC TGTAGTGCGCGTCATATTAATGCGCAGGTAGCAAAAA GTCACAACATTGCTTTGATATGGAACGTTAAAGATTTC ATGTCATTGTCTGAACAACTACGAAAACAAATACGTA GTGCTGCTAAAAAGAATAACTTACCTTTTAAGTTGACA TGTGCAACTACTAGACAAGTTGTTAATGTTGTAACAAC AAAGATAGCACTTAAGGGTGGTAAAATTGTTAATAAT TGGTTGAAGCAGTTAATTAAAGTTACACTTGTGTTCCT TTTTGTTGCTGCTATTTTCTATTTAATAACACCTGTTCA TGTCATGTCTAAACATACTGACTTTTCAAGTGAAATCA TAGGATACAAGGCTATTGATGGTGGTGTCACTCGTGA CATAGCATCTACAGATACTTGTTTTGCTAACAAACATG CTGATTTTGACACATGGTTTAGCCAGCGTGGTGGTAGT TATACTAATGACAAAGCTTGCCCATTGATTGCTGCAGT CATAACAAGAGAAGTGGGTTTTGTCGTGCCTGGTTTGC CTGGCACGATATTACGCACAACTAATGGTGACTTTTTG CATTTCTTACCTAGAGTTTTTAGTGCAGTTGGTAACAT CTGTTACACACCATCAAAACTTATAGAGTACACTGACT TTGCAACATCAGCTTGTGTTTTGGCTGCTGAATGTACA ATTTTTAAAGATGCTTCTGGTAAGCCAGTACCATATTG TTATGATACCAATGTACTAGAAGGTTCTGTTGCTTATG AAAGTTTACGCCCTGACACACGTTATGTGCTCATGGAT GGCTCTATTATTCAATTTCCTAACACCTACCTTGAAGG TTCTGTTAGAGTGGTAACAACTTTTGATTCTGAGTACT GTAGGCACGGCACTTGTGAAAGATCAGAAGCTGGTGT TTGTGTATCTACTAGTGGTAGATGGGTACTTAACAATG ATTATTACAGATCTTTACCAGGAGTTTTCTGTGGTGTA GATGCTGTAAATTTACTTACTAATATGTTTACACCACT AATTCAACCTATTGGTGCTTTGGACATATCAGCATCTA TAGTAGCTGGTGGTATTGTAGCTATCGTAGTAACATGC CTTGCCTACTATTTTATGAGGTTTAGAAGAGCTTTTGG TGAATACAGTCATGTAGTTGCCTTTAATACTTTACTAT TCCTTATGTCATTCACTGTACTCTGTTTAACACCAGTTT ACTCATTCTTACCTGGTGTTTATTCTGTTATTTACTTGT ACTTGACATTTTATCTTACTAATGATGTTTCTTTTTTAG CACATATTCAGTGGATGGTTATGTTCACACCTTTAGTA CCTTTCTGGATAACAATTGCTTATATCATTTGTATTTCC ACAAAGCATTTCTATTGGTTCTTTAGTAATTACCTAAA GAGACGTGTAGTCTTTAATGGTGTTTCCTTTAGTACTT TTGAAGAAGCTGCGCTGTGCACCTTTTTGTTAAATAAA GAAATGTATCTAAAGTTGCGTAGTGATGTGCTATTACC TCTTACGCAATATAATAGATACTTAGCTCTTTATAATA AGTACAAGTATTTTAGTGGAGCAATGGATACAACTAG CTACAGAGAAGCTGCTTGTTGTCATCTCGCAAAGGCTC TCAATGACTTCAGTAACTCAGGTTCTGATGTTCTTTAC CAACCACCACAAACCTCTATCACCTCAGCTGTTTTGCA GAGTGGTTTTAGAAAAATGGCATTCCCATCTGGTAAA GTTGAGGGTTGTATGGTACAAGTAACTTGTGGTACAA CTACACTTAACGGTCTTTGGCTTGATGACGTAGTTTAC TGTCCAAGACATGTGATCTGCACCTCTGAAGACATGCT TAACCCTAATTATGAAGATTTACTCATTCGTAAGTCTA ATCATAATTTCTTGGTACAGGCTGGTAATGTTCAACTC AGGGTTATTGGACATTCTATGCAAAATTGTGTACTTAA GCTTAAGGTTGATACAGCCAATCCTAAGACACCTAAG TATAAGTTTGTTCGCATTCAACCAGGACAGACTTTTTC AGTGTTAGCTTGTTACAATGGTTCACCATCTGGTGTTT ACCAATGTGCTATGAGGCCCAATTTCACTATTAAGGGT TCATTCCTTAATGGTTCATGTGGTAGTGTTGGTTTTAA CATAGATTATGACTGTGTCTCTTTTTGTTACATGCACC ATATGGAATTACCAACTGGAGTTCATGCTGGCACAGA CTTAGAAGGTAACTTTTATGGACCTTTTGTTGACAGGC AAACAGCACAAGCAGCTGGTACGGACACAACTATTAC AGTTAATGTTTTAGCTTGGTTGTACGCTGCTGTTATAA ATGGAGACAGGTGGTTTCTCAATCGATTTACCACAACT CTTAATGACTTTAACCTTGTGGCTATGAAGTACAATTA TGAACCTCTAACACAAGACCATGTTGACATACTAGGA CCTCTTTCTGCTCAAACTGGAATTGCCGTTTTAGATAT GTGTGCTTCATTAAAAGAATTACTGCAAAATGGTATG AATGGACGTACCATATTGGGTAGTGCTTTATTAGAAG ATGAATTTACACCTTTTGATGTTGTTAGACAATGCTCA GGTGTTACTTTCCAAAGTGCAGTGAAAAGAACAATCA AGGGTACACACCACTGGTTGTTACTCACAATTTTGACT TCACTTTTAGTTTTAGTCCAGAGTACTCAATGGTCTTT GTTCTTTTTTTTGTATGAAAATGCCTTTTTACCTTTTGC TATGGGTATTATTGCTATGTCTGCTTTTGCAATGATGT TTGTCAAACATAAGCATGCATTTCTCTGTTTGTTTTTGT TACCTTCTCTTGCCACTGTAGCTTATTTTAATATGGTCT ATATGCCTGCTAGTTGGGTGATGCGTATTATGACATGG TTGGATATGGTTGATACTAGTTTGTCTGGTTTTAAGCT AAAAGACTGTGTTATGTATGCATCAGCTGTAGTGTTAC TAATCCTTATGACAGCAAGAACTGTGTATGATGATGG TGCTAGGAGAGTGTGGACACTTATGAATGTCTTGACA CTCGTTTATAAAGTTTATTATGGTAATGCTTTAGATCA AGCCATTTCCATGTGGGCTCTTATAATCTCTGTTACTT CTAACTACTCAGGTGTAGTTACAACTGTCATGTTTTTG GCCAGAGGTATTGTTTTTATGTGTGTTGAGTATTGCCC TATTTTCTTCATAACTGGTAATACACTTCAGTGTATAA TGCTAGTTTATTGTTTCTTAGGCTATTTTTGTACTTGTT ACTTTGGCCTCTTTTGTTTACTCAACCGCTACTTTAGAC TGACTCTTGGTGTTTATGATTACTTAGTTTCTACACAG GAGTTTAGATATATGAATTCACAGGGACTACTCCCAC CCAAGAATAGCATAGATGCCTTCAAACTCAACATTAA ATTGTTGGGTGTTGGTGGCAAACCTTGTATCAAAGTAG CCACTGTACAGTCTAAAATGTCAGATGTAAAGTGCAC ATCAGTAGTCTTACTCTCAGTTTTGCAACAACTCAGAG TAGAATCATCATCTAAATTGTGGGCTCAATGTGTCCAG TTACACAATGACATTCTCTTAGCTAAAGATACTACTGA AGCCTTTGAAAAAATGGTTTCACTACTTTCTGTTTTGC TTTCCATGCAGGGTGCTGTAGACATAAACAAGCTTTGT GAAGAAATGCTGGACAACAGGGCAACCTTACAAGCTA TAGCCTCAGAGTTTAGTTCCCTTCCATCATATGCAGCT TTTGCTACTGCTCAAGAAGCTTATGAGCAGGCTGTTGC TAATGGTGATTCTGAAGTTGTTCTTAAAAAGTTGAAGA AGTCTTTGAATGTGGCTAAATCTGAATTTGACCGTGAT GCAGCCATGCAACGTAAGTTGGAAAAGATGGCTGATC AAGCTATGACCCAAATGTATAAACAGGCTAGATCTGA GGACAAGAGGGCAAAAGTTACTAGTGCTATGCAGACA ATGCTTTTCACTATGCTTAGAAAGTTGGATAATGATGC ACTCAACAACATTATCAACAATGCAAGAGATGGTTGT GTTCCCTTGAACATAATACCTCTTACAACAGCAGCCAA ACTAATGGTTGTCATACCAGACTATAACACATATAAA AATACGTGTGATGGTACAACATTTACTTATGCATCAGC ATTGTGGGAAATCCAACAGGTTGTAGATGCAGATAGT AAAATTGTTCAACTTAGTGAAATTAGTATGGACAATTC ACCTAATTTAGCATGGCCTCTTATTGTAACAGCTTTAA GGGCCAATTCTGCTGTCAAATTACAGAATAATGAGCT TAGTCCTGTTGCACTACGACAGATGTCTTGTGCTGCCG GTACTACACAAACTGCTTGCACTGATGACAATGCGTT AGCTTACTACAACACAACAAAGGGAGGTAGGTTTGTA CTTGCACTGTTATCCGATTTACAGGATTTGAAATGGGC TAGATTCCCTAAGAGTGATGGAACTGGTACTATCTATA CAGAACTGGAACCACCTTGTAGGTTTGTTACAGACAC ACCTAAAGGTCCTAAAGTGAAGTATTTATACTTTATTA AAGGATTAAACAACCTAAATAGAGGTATGGTACTTGG TAGTTTAGCTGCCACAGTACGTCTACAAGCTGGTAATG CAACAGAAGTGCCTGCCAATTCAACTGTATTATCTTTC TGTGCTTTTGCTGTAGATGCTGCTAAAGCTTACAAAGA TTATCTAGCTAGTGGGGGACAACCAATCACTAATTGT GTTAAGATGTTGTGTACACACACTGGTACTGGTCAGG CAATAACAGTTACACCGGAAGCCAATATGGATCAAGA ATCCTTTGGTGGTGCATCGTGTTGTCTGTACTGCCGTT GCCACATAGATCATCCAAATCCTAAAGGATTTTGTGA CTTAAAAGGTAAGTATGTACAAATACCTACAACTTGT GCTAATGACCCTGTGGGTTTTACACTTAAAAACACAGT CTGTACCGTCTGCGGTATGTGGAAAGGTTATGGCTGTA GTTGTGATCAACTCCGCGAACCCATGCTTCAGTCAGCT GATGCACAATCGTTTTTAAACGGGTTTGCGGTGTAAGT GCAGCCCGTCTTACACCGTGCGGCACAGGCACTAGTA CTGATGTCGTATACAGGGCTTTTGACATCTACAATGAT AAAGTAGCTGGTTTTGCTAAATTCCTAAAAACTAATTG TTGTCGCTTCCAAGAAAAGGACGAAGATGACAATTTA ATTGATTCTTACTTTGTAGTTAAGAGACACACTTTCTC TAACTACCAACATGAAGAAACAATTTATAATTTACTTA AGGATTGTCCAGCTGTTGCTAAACATGACTTCTTTAAG TTTAGAATAGACGGTGACATGGTACCACATATATCAC GTCAACGTCTTACTAAATACACAATGGCAGACCTCGT CTATGCTTTAAGGCATTTTGATGAAGGTAATTGTGACA CATTAAAAGAAATACTTGTCACATACAATTGTTGTGAT GATGATTATTTCAATAAAAAGGACTGGTATGATTTTGT AGAAAACCCAGATATATTACGCGTATACGCCAACTTA GGTGAACGTGTACGCCAAGCTTTGTTAAAAACAGTAC AATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGT GTACTGACATTAGATAATCAAGATCTCAATGGTAACT GGTATGATTTCGGTGATTTCATACAAACCACGCCAGGT AGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTT AATGCCTATATTAACCTTGACCAGGGCTTTAACTGCAG AGTCACATGTTGACACTGACTTAACAAAGCCTTACATT AAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGA GGTTAAAACTCTTTGACCGTTATTTTAAATATTGGGAT CAGACATACCACCCAAATTGTGTTAACTGTTTGGATGA CAGATGCATTCTGCATTGTGCAAACTTTAATGTTTTAT TCTCTACAGTGTTCCCACCTACAAGTTTTGGACCACTA GTGAGAAAAATATTTGTTGATGGTGTTCCATTTGTAGT TTCAACTGGATACCACTTCAGAGAGCTAGGTGTTGTAC ATAATCAGGATGTAAACTTACATAGCTCTAGACTTAGT TTTAAGGAATTACTTGTGTATGCTGCTGACCCTGCTAT GCACGCTGCTTCTGGTAATCTATTACTAGATAAACGCA CTACGTGCTTTTCAGTAGCTGCACTTACTAACAATGTT GCTTTTCAAACTGTCAAACCCGGTAATTTTAACAAAGA CTTCTATGACTTTGCTGTGTCTAAGGGTTTCTTTAAGG AAGGAAGTTCTGTTGAATTAAAACACTTCTTCTTTGCT CAGGATGGTAATGCTGCTATCAGCGATTATGACTACT ATCGTTATAATCTACCAACAATGTGTGATATCAGACA ACTACTATTTGTAGTTGAAGTTGTTGATAAGTACTTTG ATTGTTACGATGGTGGCTGTATTAATGCTAACCAAGTC ATCGTCAACAACCTAGACAAATCAGCTGGTTTTCCATT TAATAAATGGGGTAAGGCTAGACTTTATTATGATTCA ATGAGTTATGAGGATCAAGATGCACTTTTCGCATATAC AAAACGTAATGTCATCCCTACTATAACTCAAATGAAT CTTAAGTATGCCATTAGTGCAAAGAATAGAGCTCGCA CCGTAGCTGGTGTCTCTATCTGTAGTACTATGACCAAT AGACAGTTTCATCAAAAATTATTGAAATCAATAGCCG CCACTAGAGGAGCTACTGTAGTAATTGGAACAAGCAA ATTCTATGGTGGTTGGCACAACATGTTAAAAACTGTTT ATAGTGATGTAGAAAACCCTCACCTTATGGGTTGGGA TTATCCTAAATGTGATAGAGCCATGCCTAACATGCTTA GAATTATGGCCTCACTTGTTCTTGCTCGCAAACATACA ACGTGTTGTAGCTTGTCACACCGTTTCTATAGATTAGC TAATGAGTGTGCTCAAGTATTGAGTGAAATGGTCATG TGTGGCGGTTCACTATATGTTAAACCAGGTGGAACCTC ATCAGGAGATGCCACAACTGCTTATGCTAATAGTGTTT TTAACATTTGTCAAGCTGTCACGGCCAATGTTAATGCA CTTTTATCTACTGATGGTAACAAAATTGCCGATAAGTA TGTCCGCAATTTACAACACAGACTTTATGAGTGTCTCT ATAGAAATAGAGATGTTGACACAGACTTTGTGAATGA GTTTTACGCATATTTGCGTAAACATTTCTCAATGATGA TACTCTCTGACGATGCTGTTGTGTGTTTCAATAGCACT TATGCATCTCAAGGTCTAGTGGCTAGCATAAAGAACT TTAAGTCAGTTCTTTATTATCAAAACAATGTTTTTATG TCTGAAGCAAAATGTTGGACTGAGACTGACCTTACTA AAGGACCTCATGAATTTTGCTCTCAACATACAATGCTA GTTAAACAGGGTGATGATTATGTGTACCTTCCTTACCC AGATCCATCAAGAATCCTAGGGGCCGGCTGTTTTGTA GATGATATCGTAAAAACAGATGGTACACTTATGATTG AACGGTTCGTGTCTTTAGCTATAGATGCTTACCCACTT ACTAAACATCCTAATCAGGAGTATGCTGATGTCTTTCA TTTGTACTTACAATACATAAGAAAGCTACATGATGAG TTAACAGGACACATGTTAGACATGTATTCTGTTATGCT TACTAATGATAACACTTCAAGGTATTGGGAACCTGAG TTTTATGAGGCTATGTACACACCGCATACAGTCTTACA GGCTGTTGGGGCTTGTGTTCTTTGCAATTCACAGACTT CATTAAGATGTGGTGCTTGCATACGTAGACCATTCTTA TGTTGTAAATGCTGTTACGACCATGTCATATCAACATC ACATAAATTAGTCTTGTCTGTTAATCCGTATGTTTGCA ATGCTCCAGGTTGTGATGTCACAGATGTGACTCAACTT TACTTAGGAGGTATGAGCTATTATTGTAAATCACATAA ACCACCCATTAGTTTTCCATTGTGTGCTAATGGACAAG TTTTTGGTTTATATAAAAATACATGTGTTGGTAGCGAT AATGTTACTGACTTTAATGCAATTGCAACATGTGACTG GACAAATGCTGGTGATTACATTTTAGCTAACACCTGTA CTGAAAGACTCAAGCTTTTTGCAGCAGAAACGCTCAA AGCTACTGAGGAGACATTTAAACTGTCTTATGGTATTG CTACTGTACGTGAAGTGCTGTCTGACAGAGAATTACA TCTTTCATGGGAAGTTGGTAAACCTAGACCACCACTTA ACCGAAATTATGTCTTTACTGGTTATCGTGTAACTAAA AACAGTAAAGTACAAATAGGAGAGTACACCTTTGAAA AAGGTGACTATGGTGATGCTGTTGTTTACCGAGGTAC AACAACTTACAAATTAAATGTTGGTGATTATTTTGTGC TGACATCACATACAGTAATGCCATTAAGTGCACCTAC ACTAGTGCCACAAGAGCACTATGTTAGAATTACTGGC TTATACCCAACACTCAATATCTCAGATGAGTTTTCTAG CAATGTTGCAAATTATCAAAAGGTTGGTATGCAAAAG TATTCTACACTCCAGGGACCACCTGGTACTGGTAAGA GTCATTTTGCTATTGGCCTAGCTCTCTACTACCCTTCTG CTCGCATAGTGTATACAGCTTGCTCTCATGCCGCTGTT GATGCACTATGTGAGAAGGCATTAAAATATTTGCCTA TAGATAAATGTAGTAGAATTATACCTGCACGTGCTCGT GTAGAGTGTTTTGATAAATTCAAAGTGAATTCAACATT AGAACAGTATGTCTTTTGTACTGTAAATGCATTGCCTG AGACGACAGCAGATATAGTTGTCTTTGATGAAATTTC AATGGCCACAAATTATGATTTGAGTGTTGTCAATGCCA GATTACGTGCTAAGCACTATGTGTACATTGGCGACCCT GCTCAATTACCTGCACCACGCACATTGCTAACTAAGG GCACACTAGAACCAGAATATTTCAATTCAGTGTGTAG ACTTATGAAAACTATAGGTCCAGACATGTTCCTCGGA ACTTGTCGGCGTTGTCCTGCTGAAATTGTTGACACTGT GAGTGCTTTGGTTTATGATAATAAGCTTAAAGCACATA AAGACAAATCAGCTCAATGCTTTAAAATGTTTTATAA GGGTGTTATCACGCATGATGTTTCATCTGCAATTAACA GGCCACAAATAGGCGTGGTAAGAGAATTCCTTACACG TAACCCTGCTTGGAGAAAAGCTGTCTTTATTTCACCTT ATAATTCACAGAATGCTGTAGCCTCAAAGATTTTGGG ACTACCAACTCAAACTGTTGATTCATCACAGGGCTCA GAATATGACTATGTCATATTCACTCAAACCACTGAAA CAGCTCACTCTTGTAATGTAAACAGATTTAATGTTGCT ATTACCAGAGCAAAAGTAGGCATACTTTGCATAATGT CTGATAGAGACCTTTATGACAAGTTGCAATTTACAAGT CTTGAAATTCCACGTAGGAATGTGGCAACTTTACAAG CTGAAAATGTAACAGGACTCTTTAAAGATTGTAGTAA GGTAATCACTGGGTTACATCCTACACAGGCACCTACA CACCTCAGTGTTGACACTAAATTCAAAACTGAAGGTTT ATGTGTTGACATACCTGGCATACCTAAGGACATGACC TATAGAAGACTCATCTCTATGATGGGTTTTAAAATGAA TTATCAAGTTAATGGTTACCCTAACATGTTTATCACCC GCGAAGAAGCTATAAGACATGTACGTGCATGGATTGG CTTCGATGTCGAGGGGTGTCATGCTACTAGAGAAGCT GTTGGTACCAATTTACCTTTACAGCTAGGTTTTTCTAC AGGTGTTAACCTAGTTGCTGTACCTACAGGTTATGTTG ATACACCTAATAATACAGATTTTTCCAGAGTTAGTGCT AAACCACCGCCTGGAGATCAATTTAAACACCTCATAC CACTTATGTACAAAGGACTTCCTTGGAATGTAGTGCGT ATAAAGATTGTACAAATGTTAAGTGACACACTTAAAA ATCTCTCTGACAGAGTCGTATTTGTCTTATGGGCACAT GGCTTTGAGTTGACATCTATGAAGTATTTTGTGAAAAT AGGACCTGAGCGCACCTGTTGTCTATGTGATAGACGT GCCACATGCTTTTCCACTGCTTCAGACACTTATGCCTG TTGGCATCATTCTATTGGATTTGATTACGTCTATAATC CGTTTATGATTGATGTTCAACAATGGGGTTTTACAGGT AACCTACAAAGCAACCATGATCTGTATTGTCAAGTCC ATGGTAATGCACATGTAGCTAGTTGTGATGCAATCAT GACTAGGTGTCTAGCTGTCCACGAGTGCTTTGTTAAGC GTGTTGACTGGACTATTGAATATCCTATAATTGGTGAT GAACTGAAGATTAATGCGGCTTGTAGAAAGGTTCAAC ACATGGTTGTTAAAGCTGCATTATTAGCAGACAAATTC CCAGTTCTTCACGACATTGGTAACCCTAAAGCTATTAA GTGTGTACCTCAAGCTGATGTAGAATGGAAGTTCTAT GATGCACAGCCTTGTAGTGACAAAGCTTATAAAATAG AAGAATTATTCTATTCTTATGCCACACATTCTGACAAA TTCACAGATGGTGTATGCCTATTTTGGAATTGCAATGT CGATAGATATCCTGCTAATTCCATTGTTTGTAGATTTG ACACTAGAGTGCTATCTAACCTTAACTTGCCTGGTTGT GATGGTGGCAGTTTGTATGTAAATAAACATGCATTCC ACACACCAGCTTTTGATAAAAGTGCTTTTGTTAATTTA AAACAATTACCATTTTTCTATTACTCTGACAGTCCATG TGAGTCTCATGGAAAACAAGTAGTGTCAGATATAGAT TATGTACCACTAAAGTCTGCTACGTGTATAACACGTTG CAATTTAGGTGGTGCTGTCTGTAGACATCATGCTAATG AGTACAGATTGTATCTCGATGCTTATAACATGATGATC TCAGCTGGCTTTAGCTTGTGGGTTTACAAACAATTTGA TACTTATAACCTCTGGAACACTTTTACAAGACTTCAGA GTTTAGAAAATGTGGCTTTTAATGTTGTAAATAAGGG ACACTTTGATGGACAACAGGGTGAAGTACCAGTTTCT ATCATTAATAACACTGTTTACACAAAAGTTGATGGTGT TGATGTAGAATTGTTTGAAAATAAAACAACATTACCT GTTAATGTAGCATTTGAGCTTTGGGCTAAGCGCAACAT TAAACCAGTACCAGAGGTGAAAATACTCAATAATTTG GGTGTGGACATTGCTGCTAATACTGTGATCTGGGACTA CAAAAGAGATGCTCCAGCACATATATCTACTATTGGT GTTTGTTCTATGACTGACATAGCCAAGAAACCAACTG AAACGATTTGTGCACCACTCACTGTCTTTTTTGATGGT AGAGTTGATGGTCAAGTAGACTTATTTAGAAATGCCC GTAATGGTGTTCTTATTACAGAAGGTAGTGTTAAAGGT TTACAACCATCTGTAGGTCCCAAACAAGCTAGTCTTAA TGGAGTCACATTAATTGGAGAAGCCGTAAAAACACAG TTCAATTATTATAAGAAAGTTGATGGTGTTGTCCAACA ATTACCTGAAACTTACTTTACTCAGAGTAGAAATTTAC AAGAATTTAAACCCAGGAGTCAAATGGAAATTGATTT CTTAGAATTAGCTATGGATGAATTCATTGAACGGTATA AATTAGAAGGCTATGCCTTCGAACATATCGTTTATGGA GATTTTAGTCATAGTCAGTTAGGTGGTTTACATCTACT GATTGGACTAGCTAAACGTTTTAAGGAATCACCTTTTG AATTAGAAGATTTTATTCCTATGGACAGTACAGTTAAA AACTATTTCATAACAGATGCGCAAACAGGTTCATCTA AGTGTGTGTGTTCTGTTATTGATTTATTACTTGATGATT TTGTTGAAATAATAAAATCCCAAGATTTATCTGTAGTT TCTAAGGTTGTCAAAGTGACTATTGACTATACAGAAA TTTCATTTATGCTTTGGTGTAAAGATGGCCATGTAGAA ACATTTTACCCAAAATTACAATCTAGTCAAGCGTGGC AACCGGGTGTTGCTATGCCTAATCTTTACAAAATGCAA AGAATGCTATTAGAAAAGTGTGACCTTCAAAATTATG GTGATAGTGCAACATTACCTAAAGGCATAATGATGAA TGTCGCAAAATATACTCAACTGTGTCAATATTTAAACA CATTAACATTAGCTGTACCCTATAATATGAGAGTTATA CATTTTGGTGCTGGTTCTGATAAAGGAGTTGCACCAGG TACAGCTGTTTTAAGACAGTGGTTGCCTACGGGTACGC TGCTTGTCGATTCAGATCTTAATGACTTTGTCTCTGAT GCAGATTCAACTTTGATTGGTGATTGTGCAACTGTACA TACAGCTAATAAATGGGATCTCATTATTAGTGATATGT ACGACCCTAAGACTAAAAATGTTACAAAAGAAAATGA CTCTAAAGAGGGTTTTTTCACTTACATTTGTGGGTTTA TACAACAAAAGCTAGCTCTTGGAGGTTCCGTGGCTAT AAAGATAACAGAACATTCTTGGAATGCTGATCTTTAT AAGCTCATGGGACACTTCGCATGGTGGACAGCCTTTG TTACTAATGTGAATGCGTCATCATCTGAAGCATTTTTA ATTGGATGTAATTATCTTGGCAAACCACGCGAACAAA TAGATGGTTATGTCATGCATGCAAATTACATATTTTGG AGGAATACAAATCCAATTCAGTTGTCTTCCTATTCTTT ATTTGACATGAGTAAATTTCCCCTTAAATTAAGGGGTA CTGCTGTTATGTCTTTAAAAGAAGGTCAAATCAATGAT ATGATTTTATCTCTTCTTAGTAAAGGTAGACTTATAAT TAGAGAAAACAACAGAGTTGTTATTTCTAGTGATGTTC TTGTTAACAACTA S 21563 25384 ATGTTTGTTTTTCTTGTTTTATTGCCACTAGTCTCTAGT (SEQ ID CAGTGTGTTAATCTTACAACCAGAACTCAATTACCCCC NO: 4)  TGCATACACTAATTCTTTCACACGTGGTGTTTATTACC CTGACAAAGTTTTCAGATCCTCAGTTTTACATTCAACT CAGGACTTGTTCTTACCTTTCTTTTCCAATGTTACTTGG TTCCATGCTATACATGTCTCTGGGACCAATGGTACTAA GAGGTTTGATAACCCTGTCCTACCATTTAATGATGGTG TTTATTTTGCTTCCACTGAGAAGTCTAACATAATAAGA GGCTGGATTTTTGGTACTACTTTAGATTCGAAGACCCA GTCCCTACTTATTGTTAATAACGCTACTAATGTTGTTA TTAAAGTCTGTGAATTTCAATTTTGTAATGATCCATTT TTGGGTGTTTATTACCACAAAAACAACAAAAGTTGGA TGGAAAGTGAGTTCAGAGTTTATTCTAGTGCGAATAA TTGCACTTTTGAATATGTCTCTCAGCCTTTTCTTATGGA CCTTGAAGGAAAACAGGGTAATTTCAAAAATCTTAGG GAATTTGTGTTTAAGAATATTGATGGTTATTTTAAAAT ATATTCTAAGCACACGCCTATTAATTTAGTGCGTGATC TCCCTCAGGGTTTTTCGGCTTTAGAACCATTGGTAGAT TTGCCAATAGGTATTAACATCACTAGGTTTCAAACTTT ACTTGCTTTACATAGAAGTTATTTGACTCCTGGTGATT CTTCTTCAGGTTGGACAGCTGGTGCTGCAGCTTATTAT GTGGGTTATCTTCAACCTAGGACTTTTCTATTAAAATA TAATGAAAATGGAACCATTACAGATGCTGTAGACTGT GCACTTGACCCTCTCTCAGAAACAAAGTGTACGTTGA AATCCTTCACTGTAGAAAAAGGAATCTATCAAACTTCT AACTTTAGAGTCCAACCAACAGAATCTATTGTTAGATT TCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTT TTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAAC AGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTG TCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTT ATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTT ACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGA TGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAG ATTGCTGATTATAATTATAAATTACCAGATGATTTTAC AGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATT CTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTG TTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATAT TTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTA ATGGTGTTGAAGGTTTTAATTGTTACTTTCCTTTACAA TCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCA ACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTAC ATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTAC TAATTTGGTTAAAAACAAATGTGTCAATTTCAACTTCA ATGGTTTAACAGGCACAGGTGTTCTTACTGAGTCTAAC AAAAAGTTTCTGCCTTTCCAACAATTTGGCAGAGACAT TGCTGACACTACTGATGCTGTCCGTGATCCACAGACAC TTGAGATTCTTGACATTACACCATGTTCTTTTGGTGGT GTCAGTGTTATAACACCAGGAACAAATACTTCTAACC AGGTTGCTGTTCTTTATCAGGATGTTAACTGCACAGAA GTCCCTGTTGCTATTCATGCAGATCAACTTACTCCTAC TTGGCGTGTTTATTCTACAGGTTCTAATGTTTTTCAAA CACGTGCAGGCTGTTTAATAGGGGCTGAACATGTCAA CAACTCATATGAGTGTGACATACCCATTGGTGCAGGT ATATGCGCTAGTTATCAGACTCAGACTAATTCTCCTCG GCGGGCACGTAGTGTAGCTAGTCAATCCATCATTGCCT ACACTATGTCACTTGGTGCAGAAAATTCAGTTGCTTAC TCTAATAACTCTATTGCCATACCCACAAATTTTACTAT TAGTGTTACCACAGAAATTCTACCAGTGTCTATGACCA AGACATCAGTAGATTGTACAATGTACATTTGTGGTGAT TCAACTGAATGCAGCAATCTTTTGTTGCAATATGGCAG TTTTTGTACACAATTAAACCGTGCTTTAACTGGAATAG CTGTTGAACAAGACAAAAACACCCAAGAAGTTTTTGC ACAAGTCAAACAAATTTACAAAACACCACCAATTAAA GATTTTGGTGGTTTTAATTTTTCACAAATATTACCAGA TCCATCAAAACCAAGCAAGAGGTCATTTATTGAAGAT CTACTTTTCAACAAAGTGACACTTGCAGATGCTGGCTT CATCAAACAATATGGTGATTGCCTTGGTGATATTGCTG CTAGAGACCTCATTTGTGCACAAAAGTTTAACGGCCTT ACTGTTTTGCCACCTTTGCTCACAGATGAAATGATTGC TCAATACACTTCTGCACTGTTAGCGGGTACAATCACTT CTGGTTGGACCTTTGGTGCAGGTGCTGCATTACAAATA CCATTTGCTATGCAAATGGCTTATAGGTTTAATGGTAT TGGAGTTACACAGAATGTTCTCTATGAGAACCAAAAA TTGATTGCCAACCAATTTAATAGTGCTATTGGCAAAAT TCAAGACTCACTTTCTTCCACAGCAAGTGCACTTGGAA AACTTCAAGATGTGGTCAACCAAAATGCACAAGCTTT AAACACGCTTGTTAAACAACTTAGCTCCAATTTTGGTG CAATTTCAAGTGTTTTAAATGATATCCTTTCACGTCTT GACAAAGTTGAGGCTGAAGTGCAAATTGATAGGTTGA TCACAGGCAGACTTCAAAGTTTGCAGACATATGTGAC TCAACAATTAATTAGAGCTGCAGAAATCAGAGCTTCT GCTAATCTTGCTGCTACTAAAATGTCAGAGTGTGTACT TGGACAATCAAAAAGAGTTGATTTTTGTGGAAAGGGC TATCATCTTATGTCCTTCCCTCAGTCAGCACCTCATGG TGTAGTCTTCTTGCATGTGACTTATGTCCCTGCACAAG AAAAGAACTTCACAACTGCTCCTGCCATTTGTCATGAT GGAAAAGCACACTTTCCTCGTGAAGGTGTCTTTGTTTC AAATGGCACACACTGGTTTGTAACACAAAGGAATTTT TATGAACCACAAATCATTACTACAGACAACACATTTG TGTCTGGTAACTGTGATGTTGTAATAGGAATTGTCAAC AACACAGTTTATGATCCTTTGCAACCTGAATTAGACTC ATTCAAGGAGGAGTTAGATAAATATTTTAAGAATCAT ACATCACCAGATGTTGATTTAGGTGACATCTCTGGCAT TAATGCTTCAGTTGTAAACATTCAAAAAGAAATTGAC CGCCTCAATGAGGTTGCCAAGAATTTAAATGAATCTCT CATCGATCTCCAAGAACTTGGAAAGTATGAGCAGTAT ATAAAATGGCCATGGTACATTTGGCTAGGTTTTATAGC TGGCTTGATTGCCATAGTAATGGTGACAATTATGCTTT GCTGTATGACCAGTTGCTGTAGTTGTCTCAAGGGCTGT TGTTCTTGTGGATCCTGCTGCAAATTTGATGAAGACGA CTCTGAGCCAGTGCTCAAAGGAGTCAAATTACATTAC ACATA 3a 25393 26220 ATGGATTTGTTTATGAGAATCTTCACAATTGGAACTGT (SEQ ID  AACTTTGAAGCAAGGTGAAATCAAGGATGCTACTCCT NO: 5) TCAGATTTTGTTCGCGCTACTGCAACGATACCGATACA AGCCTCACTCCCTTTCGGATGGCTTATTGTTGGCGTTG CACTTCTTGCTGTTTTTCAGAGCGCTTCCAAAATCATA ACCCTCAAAAAGAGATGGCAACTAGCACTCTCCAAGG GTGTTCACTTTGTTTGCAACTTGCTGTTGTTGTTTGTAA CAGTTTACTCACACCTTTTGCTCGTTGCTGCTGGCCTT GAAGCCCCTTTTCTCTATCTTTATGCTTTAGTCTACTTC TTGCAGAGTATAAACTTTGTAAGAATAATAATGAGGC TTTGGCTTTGCTGGAAATGCCGTTCCAAAAACCCATTA CTTTATGATGCCAACTATTTTCTTTGCTGGCATACTAA TTGTTACGACTATTGTATACCTTACAATAGTGTAACTT CTTCAATTGTCATTACTTCAGGTGATGGCACAACAAGT CCTATTTCTGAACATGACTACCAGATTGGTGGTTATAC TGAAAAATGGGAATCTGGAGTAAAAGACTGTGTTGTA TTACACAGTTACTTCACTTCAGACTATTACCAGCTGTA CTCAACTCAATTGAGTACAGACACTGGTGTTGAACAT GTTACCTTCTTCATCTACAATAAAATTGTTGATGAGCC TGAAGAACATGTCCAAATTCACACAATCGACGGTTCA TCCGGAGTTGTTAATCCAGTAATGGAACCAATTTATGA TGAACCGACGACGACTACTAGCGTGCCTTTGTA E 26245 26472 ATGTACTCATTCGTTTCGGAAGAGACAGGTACGTTAAT (SEQ ID  AGTTAATAGCGTACTTCTTTTTCTTGCTTTCGTGGTATT NO: 6) CTTGCTAGTTACACTAGCCATCCTTACTGCGCTTCGAT TGTGTGCGTACTGCTGCAATATTGTTAACGTGAGTCTT GTAAAACCTTCTTTTTACGTTTACTCTCGTGTTAAAAA TCTGAATTCTTCTAGAGTTCCTGATCTTCTGGTCTA M 26523 27191 ATGGCAGATTCCAACGGTACTATTACCGTTGAAGAGC (SEQ ID  TTAAAAAGCTCCTTGAACAATGGAACCTAGTAATAGG NO: 7) TTTCCTATTCCTTACATGGATTTGTCTTCTACAATTTGC CTATGCCAACAGGAATAGGTTTTTGTATATAATTAAGT TAATTTTCCTCTGGCTGTTATGGCCAGTAACTTTAGCT TGTTTTGTGCTTGCTGCTGTTTACAGAATAAATTGGAT CACCGGTGGAATTGCTATCGCAATGGCTTGTCTTGTAG GCTTGATGTGGCTCAGCTACTTCATTGCTTCTTTCAGA CTGTTTGCGCGTACGCGTTCCATGTGGTCATTCAATCC AGAAACTAACATTCTTCTCAACGTGCCACTCCATGGCA CTATTCTGACCAGACCGCTTCTAGAAAGTGAACTCGTA ATCGGAGCTGTGATCCTTCGTGGACATCTTCGTATTGC TGGACACCATCTAGGACGCTGTGACATCAAGGACCTG CCTAAAGAAATCACTGTTGCTACATCACGAACGCTTTC TTATTACAAATTGGGAGCTTCGCAGCGTGTAGCAGGT GACTCAGGTTTTGCTGCATACAGTCGCTACAGGATTGG CAACTATAAATTAAACACAGACCATTCCAGTAGCAGT GACAATATTGCTTTGCTTGTACAGTA 7a 27394 27759 ATGAAAATTATTCTTTTCTTGGCACTGATAACACTCGC (SEQ ID  TACTTGTGAGCTTTATCACTACCAAGAGTGTGTTAGAG NO: 8) GTACAACAGTACTTTTAAAAGAACCTTGCTCTTCTGGA ACATACGAGGGCAATTCACCATTTCATCCTCTAGCTGA TAACAAATTTGCACTGACTTGCTTTAGCACTCAATTTG CTTTTGCTTGTCCTGACGGCGTAAAACACGTCTATCAG TTACGTGCCAGATCAGTTTCACCTAAACTGTTCATCAG ACAAGAGGAAGTTCAAGAACTTTACTCTCCAATTTTTC TTATTGTTGCGGCAATAGTGTTTATAACACTTTGCTTC ACACTCAAAAGAAAGACAGAATG 8a 27894 28259 ATGAAATTTCTTGTTTTCTTAGGAATCATCACAACTGT (SEQ ID  AGCTGCATTTCACCAAGAATGTAGTTTACAGTCATGTA NO: 9) CTCAACATCAACCATATGTAGTTGATGACCCGTGTCCT ATTCACTTCTATTCTAAATGGTATATTAGAGTAGGAGC TAGAAAATCAGCACCTTTAATTGAATTGTGCGTGGAT GAGGCTGGTTCTAAATCACCCATTCAGTACATCGATAT CGGTAATTATACAGTTTCCTGTTTACCTTTTACAATTA ATTGCCAGGAACCTAAATTGGGTAGTCTTGTAGTGCGT TGTTCGTTCTATGAAGACTTTTTAGAGTATCATGACGT TCGTGTTGTTTTAGATTTCATCTA N 28274 29533 ATGTCTGATAATGGACCCCAAAATCAGCGAAATGCAC (SEQ ID  CCCGCATTACGTTTGGTGGACCCTCAGATTCAACTGGC NO: 10) AGTAACCAGAATGGAGAACGCAGTGGGGCGCGATCA AAACAACGTCGGCCCCAAGGTTTACCCAATAATACTG CGTCTTGGTTCACCGCTCTCACTCAACATGGCAAGGAA GACCTTAAATTCCCTCGAGGACAAGGCGTTCCAATTA ACACCAATAGCAGTCCAGATGACCAAATTGGCTACTA CCGAAGAGCTACCAGACGAATTCGTGGTGGTGACGGT AAAATGAAAGATCTCAGTCCAAGATGGTATTTCTACT ACCTAGGAACTGGGCCAGAAGCTGGACTTCCCTATGG TGCTAACAAAGACGGCATCATATGGGTTGCAACTGAG GGAGCCTTGAATACACCAAAAGATCACATTGGCACCC GCAATCCTGCTAACAATGCTGCAATCGTGCTACAACTT CCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAG AAGGGAGCAGAGGCGGCAGTCAAGCCTCTTCTCGTTC CTCATCACGTAGTCGCAACAGTTCAAGAAATTCAACT CCAGGCAGCAGTAGGGGAACTTCTCCTGCTAGAATGG CTGGCAATGGCGGTGATGCTGCTCTTGCTTTGCTGCTG CTTGACAGATTGAACCAGCTTGAGAGCAAAATGTCTG GTAAAGGCCAACAACAACAAGGCCAAACTGTCACTAA GAAATCTGCTGCTGAGGCTTCTAAGAAGCCTCGGCAA AAACGTACTGCCACTAAAGCATACAATGTAACACAAG CTTTCGGCAGACGTGGTCCAGAACAAACCCAAGGAAA TTTTGGGGACCAGGAACTAATCAGACAAGGAACTGAT TACAAACATTGGCCGCAAATTGCACAATTTGCCCCCA GCGCTTCAGCGTTCTTCGGAATGTCGCGCATTGGCATG GAAGTCACACCTTCGGGAACGTGGTTGACCTACACAG GTGCCATCAAATTGGATGACAAAGATCCAAATTTCAA AGATCAAGTCATTTTGCTGAATAAGCATATTGACGCAT ACAAAACATTCCCACCAACAGAGCCTAAAAAGGACAA AAAGAAGAAGGCTGATGAAACTCAAGCCTTACCGCAG AGACAGAAGAAACAGCAAACTGTGACTCTTCTTCCTG CTGCAGATTTGGATGATTTCTCCAAACAATTGCAACAA TCCATGAGCAGTGCTGACTCAACTCAGGCCTA

The invention covers a combination of bioinformatic approaches to identify regions of conservation (based on 718 patient isolates) in combination with features essential for RISC entry and tolerance of chemical modifications. The method uses a scoring scheme to identify siRNAs that target regions of related viral genomes with low mutation rates. Since viral targets are known to mutate frequently, it was imperative to select siRNAs targeting regions that are predicted to remain constant. We identified these regions as those with high homology to the six most closely-related coronavirus genomes (Middle East respiratory syndrome-related coronavirus (MERS-CoV), Human coronavirus 229E, Human coronavirus NL63 (HCoV-NL63), Human coronavirus HKU1 (HCoV-HKU1), Human coronavirus OC43 (HCoV-OC43), and SARS coronavirus) indicating low rates of mutation within these regions.

The percent homology to the related coronavirus genomes was determined for every position for each of the SARS-CoV-2 target genes. siRNA sequence designs were then scored by the number of positions within the sequence having a percentage homology greater than 70% within position 2-8 and greater than 50% for at least 10 bases within the remaining positions of the 16-nucleotide targeting region of the 20-nucleotide siRNA. The design algorithm identified a 20-nucleotide siRNA sequences and scored them by their predicted efficiency to knockdown the target transcript. The 20-nucleotide siRNA target regions are summarized in Table 6A and Table 7. Top scoring siRNAs had the highest potential to knockdown the target transcript and targeted regions with the highest homology to other closely related coronaviruses (Middle East respiratory syndrome-related coronavirus (MERS-CoV), Human coronavirus 229E, Human coronavirus NL63 (HCoV-NL63), Human coronavirus HKU1 (HCoV-HKU1), Human coronavirus OC43 (HCoV-OC43), and SARS coronavirus) are summarized in Table 8 and were selected for synthesis. ASOs targeting SARS-CoV-2 genes are summarized in Table 9.

TABLE 6A SARS-CoV2 Screened-20 nucleotide target sequences and 45 nucleotide target gene regions SEQ SEQ ID ID Sequence ID 20 nt Sequence NO: 45 nt Gene Region NO: orf1ab_14080 GGUAACUGG  11 AATCAAGATCTCAATGGTAACTGGTAT 119 UAUGAUUUC GATTTCGGTGATTTCATA GG orf1ab_14361 UCUGCAUUG  12 GGATGACAGATGCATTCTGCATTGTGC 120 UGCAAACUU AAACTTTAATGTTTTATT UA orf1ab_14830 UGUGAUAUC  13 AATCTACCAACAATGTGTGATATCAGA 121 AGACAACUA CAACTACTATTTGTAGTT CU orf1ab_15376 UGUAGCUUG  14 AAACATACAACGTGTTGTAGCTTGTCA 122 UCACACCGU CACCGTTTCTATAGATTA UU orf1ab_15786 UAAGUCAGU  15 TAGCATAAAGAACTTTAAGTCAGTTCT 123 UCUUUAUUA TTATTATCAAAACAATGT UC orf1ab_17107 UUUGCUAUU  16 ACTGGTAAGAGTCATTTTGCTATTGGC 124 GGCCUAGCU CTAGCTCTCTACTACCCT CU orf1ab_17370 GGCCACAAA  17 TGATGAAATTTCAATGGCCACAAATTA 125 UUAUGAUU TGATTTGAGTGTTGTCAA UGA orf1ab_18025 GUGGCAACU  18 ATTCCACGTAGGAATGTGGCAACTTTA 126 UUACAAGCU CAAGCTGAAAATGTAACA GA orf1ab_18571 UCUGACAGA  19 ACACTTAAAAATCTCTCTGACAGAGTC 127 GUCGUAUUU GTATTTGTCTTATGGGCA GU orf1ab_20497 UCUGUUAUU  20 TCTAAGTGTGTGTGTTCTGTTATTGATT 128 GAUUUAUU TATTACTTGATGATTTT ACU orf1ab_20892 UGCACCAGG  21 TTCTGATAAAGGAGTTGCACCAGGTAC 129 UACAGCUGU AGCTGTTTTAAGACAGTG UU orf1ab_21391 UUUGACAUG  22 TCTTCCTATTCTTTATTTGACATGAGTA 130 AGUAAAUU AATTTCCCCTTAAATTA UCC orf1a_416 UGUGGCUUA  23 CTTAAAGATGGCACTTGTGGCTTAGTA 131 GUAGAAGU GAAGTTGAAAAAGGCGTT UGA orf1a_2290 UCAGACAUU  24 AATTAAGGAGAGTGTTCAGACATTCTT 132 CUUUAAGCU TAAGCTTGTAAATAAATT UG orf1a_6059 UGUGAUAA  25 AATTTTAAGTTTGTATGTGATAATATC 133 UAUCAAAUU AAATTTGCTGATGATTTA UGC orf1a_6322 UGAAACAUC  26 GAGCACAAAACCAGTTGAAACATCAA 134 AAAUUCGUU ATTCGTTTGATGTACTGAA UG orf1a_6499 ACCAGCAAA  27 AGACATTATACTTAAACCAGCAAATAA 135 UAAUAGUU TAGTTTAAAAATTACAGA UAA orf1a_7643 GCUGGUAGU  28 TGTGATACATTCTGTGCTGGTAGTACA 136 ACAUUUAUU TTTATTAGTGATGAAGTT AG orf1a_8200 UGUAGAAAC  29 GTTTGTTGATTCAGATGTAGAAACTAA 137 UAAAGAUG AGATGTTGTTGAATGTCT UUG orf1a_8201 GUAGAAACU  30 TTTGTTGATTCAGATGTAGAAACTAAA 138 AAAGAUGU GATGTTGTTGAATGTCTT UGU orf1a_8744 UUUGCUAAC  31 TCTACAGATACTTGTTTTGCTAACAAA 139 AAACAUGCU CATGCTGATTTTGACACA GA orf1a_9679 CUGGAUAAC  32 ACCTTTAGTACCTTTCTGGATAACAAT 140 AAUUGCUUA TGCTTATATCATTTGTAT UA orf1a_11594 CAGUGUAUA  33 ACTGGTAATACACTTCAGTGTATAATG 141 AUGCUAGUU CTAGTTTATTGTTTCTTA UA orf1a_12932 CCUAAAGUG  34 GACACACCTAAAGGTCCTAAAGTGAA 142 AAGUAUUU GTATTTATACTTTATTAAA AUA S_21944 AUUAAAGUC  35 GCTACTAATGTTGTTATTAAAGTCTGT 143 UGUGAAUU GAATTTCAATTTTGTAAT UCA S_22223 UCGGCUUUA  36 CTCCCTCAGGGTTTTTCGGCTTTAGAA 144 GAACCAUUG CCATTGGTAGATTTGCCA GU S_22550 CCUAAUAUU  37 TCTATTGTTAGATTTCCTAATATTACAA 145 ACAAACUUG ACTTGTGCCCTTTTGGT UG S_22820 GAUUAUAA  38 ACTGGAAAGATTGCTGATTATAATTAT 146 UUAUAAAU AAATTACCAGATGATTTT UACC S_22898 GGUGGUAA  39 CTTGATTCTAAGGTTGGTGGTAATTAT 147 UUAUAAUU AATTACCTGTATAGATTG ACCU S_23174 UGUGUCAAU  40 TTGGTTAAAAACAAATGTGTCAATTTC 148 UUCAACUUC AACTTCAATGGTTTAACA AA S_23239 UCUGCCUUU  41 GTCTAACAAAAAGTTTCTGCCTTTCCA 149 CCAACAAUU ACAATTTGGCAGAGACAT UG S_23240 CUGCCUUUC  42 TCTAACAAAAAGTTTCTGCCTTTCCAA 150 CAACAAUUU CAATTTGGCAGAGACATT GG S_23774 UGUACAAUG  43 AAGACATCAGTAGATTGTACAATGTAC 151 UACAUUUGU ATTTGTGGTGATTCAACT GG S_24056 GGCUUCAUC  44 ACACTTGCAGATGCTGGCTTCATCAAA 152 AAACAAUAU CAATATGGTGATTGCCTT GG S_24289 UGGAGUUAC  45 TAGGTTTAATGGTATTGGAGTTACACA 153 ACAGAAUGU GAATGTTCTCTATGAGAA UC S_25375 UUACACAUA  46 AGGAGTCAAATTACATTACACATAAAC 154 AACGAACUU GAACTTATGGATTTGTTT AU 3a_25413 CUUCACAAU  47 TTTGTTTATGAGAATCTTCACAATTGG 155 UGGAACUGU AACTGTAACTTTGAAGCA AA 3a_25630 GUUUGCAAC  48 AAGGGTGTTCACTTTGTTTGCAACTTG 156 UUGCUGUUG CTGTTGTTGTTTGTAACA UU 3a_25717 UAUGCUUUA  49 CCTTTTCTCTATCTTTATGCTTTAGTCT 157 GUCUACUUC ACTTCTTGCAGAGTATA UU 3a_25734 CUUGCAGAG  50 TGCTTTAGTCTACTTCTTGCAGAGTAT 158 UAUAAACUU AAACTTTGTAAGAATAAT UG 3a_25736 UGCAGAGUA  51 CTTTAGTCTACTTCTTGCAGAGTATAA 159 UAAACUUUG ACTTTGTAAGAATAATAA UA 3a_25745 UAAACUUUG  52 ACTTCTTGCAGAGTATAAACTTTGTAA 160 UAAGAAUA GAATAATAATGAGGCTTT AUA 3a_25868 CUUACAAUA  53 ACGACTATTGTATACCTTACAATAGTG 161 GUGUAACUU TAACTTCTTCAATTGTCA CU 3a_25870 UACAAUAGU  54 GACTATTGTATACCTTACAATAGTGTA 162 GUAACUUCU ACTTCTTCAATTGTCATT UC 3a_25914 CACAACAAG  55 TACTTCAGGTGATGGCACAACAAGTCC 163 UCCUAUUUC TATTTCTGAACATGACTA UG 3a_25992 UGUUGUAU  56 TGGAGTAAAAGACTGTGTTGTATTACA 164 UACACAGUU CAGTTACTTCACTTCAGA ACU 3a_26018 CAGACUAUU  57 ACAGTTACTTCACTTCAGACTATTACC 165 ACCAGCUGU AGCTGTACTCAACTCAAT AC 3a_26066 UUGAACAUG  58 GTACAGACACTGGTGTTGAACATGTTA 166 UUACCUUCU CCTTCTTCATCTACAATA UC E_26258 UUUCGGAAG  59 TTATGTACTCATTCGTTTCGGAAGAGA 167 AGACAGGUA CAGGTACGTTAATAGTTA CG E_26261 CGGAAGAGA  60 TGTACTCATTCGTTTCGGAAGAGACAG 168 CAGGUACGU GTACGTTAATAGTTAATA UA E_26269 ACAGGUACG  61 TTCGTTTCGGAAGAGACAGGTACGTTA 169 UUAAUAGU ATAGTTAATAGCGTACTT UAA E_26277 GUUAAUAG  62 GGAAGAGACAGGTACGTTAATAGTTA 170 UUAAUAGCG ATAGCGTACTTCTTTTTCT UAC E_26305 CUUGCUUUC  63 AGCGTACTTCTTTTTCTTGCTTTCGTGG 171 GUGGUAUUC TATTCTTGCTAGTTACA UU E_26313 CGUGGUAUU  64 TCTTTTTCTTGCTTTCGTGGTATTCTTG 172 CUUGCUAGU CTAGTTACACTAGCCAT UA E_26369 ACUGCUGCA  65 TTCGATTGTGTGCGTACTGCTGCAATA 173 AUAUUGUU TTGTTAACGTGAGTCTTG AAC E_26374 UGCAAUAUU  66 TTGTGTGCGTACTGCTGCAATATTGTT 174 GUUAACGUG AACGTGAGTCTTGTAAAA AG E_26455 CCUGAUCUU  67 AATTCTTCTAGAGTTCCTGATCTTCTGG 175 CUGGUCUAA TCTAAACGAACTAAATA AC E_26463 UCUGGUCUA  68 TAGAGTTCCTGATCTTCTGGTCTAAAC 176 AACGAACUA GAACTAAATATTATATTA AA E_26467 GUCUAAACG  69 GTTCCTGATCTTCTGGTCTAAACGAAC 177 AACUAAAUA TAAATATTATATTAGTTT UU E_26470 UAAACGAAC  70 CCTGATCTTCTGGTCTAAACGAACTAA 178 UAAAUAUU ATATTATATTAGTTTTTC AUA M_26573 UGAACAAUG  71 GCTTAAAAAGCTCCTTGAACAATGGAA 179 GAACCUAGU CCTAGTAATAGGTTTCCT AA M_26581 GGAACCUAG  72 AGCTCCTTGAACAATGGAACCTAGTAA 180 UAAUAGGU TAGGTTTCCTATTCCTTA UUC M_26602 UAUUCCUUA  73 TAGTAATAGGTTTCCTATTCCTTACAT 181 CAUGGAUUU GGATTTGTCTTCTACAAT GU M_26624 UCUACAAUU  74 TACATGGATTTGTCTTCTACAATTTGCC 182 UGCCUAUGC TATGCCAACAGGAATAG CA M_26637 UAUGCCAAC  75 CTTCTACAATTTGCCTATGCCAACAGG 183 AGGAAUAG AATAGGTTTTTGTATATA GUU M_26638 AUGCCAACA  76 TTCTACAATTTGCCTATGCCAACAGGA 184 GGAAUAGG ATAGGTTTTTGTATATAA UUU M_26693 AUGGCCAGU  77 TTTCCTCTGGCTGTTATGGCCAGTAAC 185 AACUUUAGC TTTAGCTTGTTTTGTGCT UU M_26717 UGUGCUUGC  78 AACTTTAGCTTGTTTTGTGCTTGCTGCT 186 UGCUGUUUA GTTTACAGAATAAATTG CA M_27014 GCCUAAAGA  79 TGACATCAAGGACCTGCCTAAAGAAA 187 AAUCACUGU TCACTGTTGCTACATCACG UG M_27032 UGCUACAUC  80 TAAAGAAATCACTGTTGCTACATCACG 188 ACGAACGCU AACGCTTTCTTATTACAA UU M_27035 UACAUCACG  81 AGAAATCACTGTTGCTACATCACGAAC 189 AACGCUUUC GCTTTCTTATTACAAATT UU M_27123 AUUGGCAAC  82 TACAGTCGCTACAGGATTGGCAACTAT 190 UAUAAAUU AAATTAAACACAGACCAT AAA 7a_27455 AAGAGUGU  83 AGCTTTATCACTACCAAGAGTGTGTTA 191 GUUAGAGG GAGGTACAACAGTACTTT UACA 7a_27522 UUCACCAUU  84 AACATACGAGGGCAATTCACCATTTCA 192 UCAUCCUCU TCCTCTAGCTGATAACAA AG 7a_27537 UCUAGCUGA  85 TTCACCATTTCATCCTCTAGCTGATAA 193 UAACAAAUU CAAATTTGCACTGACTTG UG 7a_27553 UUUGCACUG  86 CTAGCTGATAACAAATTTGCACTGACT 194 ACUUGCUUU TGCTTTAGCACTCAATTT AG 7a_27565 UGCUUUAGC  87 AAATTTGCACTGACTTGCTTTAGCACT 195 ACUCAAUUU CAATTTGCTTTTGCTTGT GC 7a_27633 AUCAGUUUC  88 TCAGTTACGTGCCAGATCAGTTTCACC 196 ACCUAAACU TAAACTGTTCATCAGACA GU 7a_27656 UCAGACAAG  89 CACCTAAACTGTTCATCAGACAAGAGG 197 AGGAAGUUC AAGTTCAAGAACTTTACT AA 7a_27671 UUCAAGAAC  90 TCAGACAAGAGGAAGTTCAAGAACTT 198 UUUACUCUC TACTCTCCAATTTTTCTTA CA 7a_27705 UGCGGCAAU  91 AATTTTTCTTATTGTTGCGGCAATAGT 199 AGUGUUUA GTTTATAACACTTTGCTT UAA 7a_27715 GUGUUUAU  92 ATTGTTGCGGCAATAGTGTTTATAACA 200 AACACUUUG CTTTGCTTCACACTCAAA CUU 7a_27720 UAUAACACU  93 TGCGGCAATAGTGTTTATAACACTTTG 201 UUGCUUCAC CTTCACACTCAAAAGAAA AC 7a_27751 ACAGAAUGA  94 ACACTCAAAAGAAAGACAGAATGATT 202 UUGAACUUU GAACTTTCATTAATTGACT CA 8b_27932 AGCUGCAUU  95 AATCATCACAACTGTAGCTGCATTTCA 203 UCACCAAGA CCAAGAATGTAGTTTACA AU 8b_27940 UUCACCAAG  96 CAACTGTAGCTGCATTTCACCAAGAAT 204 AAUGUAGU GTAGTTTACAGTCATGTA UUA 8b_27986 UGUAGUUG  97 TCAACATCAACCATATGTAGTTGATGA 205 AUGACCCGU CCCGTGTCCTATTCACTT GUC 8b_28002 UGUCCUAUU  98 GTAGTTGATGACCCGTGTCCTATTCAC 206 CACUUCUAU TTCTATTCTAAATGGTAT UC 8b_28024 AAUGGUAU  99 TTCACTTCTATTCTAAATGGTATATTAG 207 AUUAGAGU AGTAGGAGCTAGAAAAT AGGA 8b_28091 UUCUAAAUC 100 CGTGGATGAGGCTGGTTCTAAATCACC 208 ACCCAUUCA CATTCAGTACATCGATAT GU 8b_28119 AUCGGUAAU 101 ATTCAGTACATCGATATCGGTAATTAT 209 UAUACAGUU ACAGTTTCCTGTTTACCT UC 8b_28127 UUAUACAGU 102 CATCGATATCGGTAATTATACAGTTTC 210 UUCCUGUUU CTGTTTACCTTTTACAAT AC 8b_28128 UAUACAGUU 103 ATCGATATCGGTAATTATACAGTTTCC 211 UCCUGUUUA TGTTTACCTTTTACAATT CC 8b_28163 CCAGGAACC 104 TTTTACAATTAATTGCCAGGAACCTAA 212 UAAAUUGG ATTGGGTAGTCTTGTAGT GUA 8b_28218 UUAGAGUA 105 TTCTATGAAGACTTTTTAGAGTATCAT 213 UCAUGACGU GACGTTCGTGTTGTTTTA UCG 8b_28222 AGUAUCAUG 106 ATGAAGACTTTTTAGAGTATCATGACG 214 ACGUUCGUG TTCGTGTTGTTTTAGATT UU N_28407 UACCCAAUA 107 GTCGGCCCCAAGGTTTACCCAATAATA 215 AUACUGCGU CTGCGTCTTGGTTCACCG CU N_28655 GACGGCAUC 108 TATGGTGCTAACAAAGACGGCATCATA 216 AUAUGGGU TGGGTTGCAACTGAGGGA UGC N_28945 UGACAGAUU 109 TGCTTTGCTGCTGCTTGACAGATTGAA 217 GAACCAGCU CCAGCTTGAGAGCAAAAT UG N_28992 AACAACAAG 110 CTGGTAAAGGCCAACAACAACAAGGC 218 GCCAAACUG CAAACTGTCACTAAGAAAT UC N_29141 GAACUAAUC 111 AATTTTGGGGACCAGGAACTAATCAG 219 AGACAAGGA ACAAGGAACTGATTACAAA AC N_29276 GGUGCCAUC 112 TGGTTGACCTACACAGGTGCCATCAAA 220 AAAUUGGA TTGGATGACAAAGATCCA UGA N_29292 AUGACAAAG 113 GTGCCATCAAATTGGATGACAAAGATC 221 AUCCAAAUU CAAATTTCAAAGATCAAG UC N_29293 UGACAAAGA 114 TGCCATCAAATTGGATGACAAAGATCC 222 UCCAAAUUU AAATTTCAAAGATCAAGT CA N_29303 CCAAAUUUC 115 TTGGATGACAAAGATCCAAATTTCAAA 223 AAAGAUCAA GATCAAGTCATTTTGCTG GU N_29307 AUUUCAAAG 116 ATGACAAAGATCCAAATTTCAAAGATC 224 AUCAAGUCA AAGTCATTTTGCTGAATA UU N_29328 UGCUGAAUA 117 AAGATCAAGTCATTTTGCTGAATAAGC 225 AGCAUAUUG ATATTGACGCATACAAAA AC N_29464 UGCAGAUUU 118 GACTCTTCTTCCTGCTGCAGATTTGGA 226 GGAUGAUU TGATTTCTCCAAACAATT UCU

TABLE 6B Modified antisense strand (21 nucleotide length for screening) Sequence SEQ ID Modified AS strand (21mer for screening) ID NO: orf1ab_14080 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_14361 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_14830 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_15376 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_15786 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_17107 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_17370 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_18025 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_18571 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_20497 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_20892 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1ab_21391 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_416 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_2290 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_6059 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_6322 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_6499 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_7643 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_8200 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_8201 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_8744 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_9679 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_11594 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 orf1a_12932 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_21944 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_22223 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_22550 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_22820 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_22898 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_23174 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_23239 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_23240 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_23774 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_24056 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_24289 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 S_25375 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25413 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25630 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25717 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25734 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25736 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25745 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25868 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25870 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25914 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_25992 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_26018 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 3a_26066 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26258 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26261 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26269 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26277 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26305 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26313 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26369 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26374 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26455 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26463 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26467 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 E_26470 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26573 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26581 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26602 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26624 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26637 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26638 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26693 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_26717 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_27014 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_27032 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_27035 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 M_27123 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27455 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27522 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27537 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27553 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27565 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27633 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27656 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27671 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27705 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27715 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27720 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 7a_27751 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_27932 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_27940 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_27986 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28002 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28024 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28091 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28119 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28127 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28128 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28163 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28218 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 8b_28222 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_28407 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_28655 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_28945 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_28992 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29141 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29276 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29292 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29293 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29303 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29307 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29328 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227 N_29464 P(mU)#(fA)#(mA)(mC)(mA)(fU)(mG)(mA)(mA)(mU)(mA)(mC)(mU)(fU)#(mG)#(fG)#(mC)#(mU)#(mU)#(mU)#(mU) 227

TABLE 6C Modified sense strand - 16 nucleotides in length Sequence SEQ ID ID Modified sense strand 16 nt NO: orf1ab_14080 (mA)#(mU)#(mC)(mA)(fU)(fA)(fC)(mC)(fA)(mG)(mU)(mU)(mA)#(mC)#(mA)-TegChol 228 orf1ab_14361 (mG)#(mU)#(mU)(mU)(mG)(fC)(fA)(fC)(mA)(fA)(mU)(mG)(mC)(mA)#(mG)#(mA)-TegChol 229 orf1ab_14830 (mU)#(mU)#(mU)(mG)(fC)(fA)(fC)(mA)(fA)(mU)(mG)(mC)(mA)#(mG)#(mA)-TegChol 230 orf1ab_15376 (mG)#(mU)#(mG)(mU)(fG)(fA)(fC)(mA)(fA)(mG)(mC)(mU)(mA)#(mC)#(mA)-TegChol 231 orf1ab_15786 (mU)#(mA)#(mA)(mA)(fG)(fA)(fA)(mC)(fU)(mG)(mA)(mC)(mU)#(mU)#(mA)-TegChol 232 orf1ab_17107 (mU)#(mA)#(mG)(mG)(fC)(fC)(fA)(mA)(fU)(mA)(mG)(mC)(mA)#(mA)#(mA)-TegChol 233 orf1ab_17370 (mU)#(mC)#(mA)(mU)(fA)(fA)(fU)(mU)(fU)(mG)(mU)(mG)(mG)#(mC)#(mA)-TegChol 234 orf1ab_18025 (mU)#(mU)#(mG)(mU)(fA)(fA)(fA)(mG)(fU)(mU)(mG)(mC)(mC)#(mA)#(mA)-TegChol 235 orf1ab_18571 (mU)#(mA)#(mC)(mG)(fA)(fC)(fU)(mC)(fU)(mG)(mU)(mC)(mA)#(mG)#(mA)-TegChol 236 orf1ab_20497 (mU)#(mA)#(mA)(mA)(fU)(fC)(fA)(mA)(fU)(mA)(mA)(mC)(mA)#(mG)#(mA)-TegChol 237 orf1ab_20892 (mG)#(mC)#(mU)(mG)(fU)(fA)(fC)(mC)(fU)(mG)(mG)(mU)(mG)#(mC)#(mA)-TegChol 238 orf1ab_21391 (mU)#(mU)#(mU)(mA)(fC)(fU)(fC)(mA)(fU)(mG)(mU)(mC)(mA)#(mA)#(mA)-TegChol 239 orf1a_416 (mU)#(mU)#(mC)(mU)(fA)(fC)(fU)(mA)(fA)(mG)(mC)(mC)(mA)#(mC)#(mA)-TegChol 240 orf1a_2290 (mU)#(mU)#(mA)(mA)(fA)(fG)(fA)(mA)(fU)(mG)(mU)(mC)(mU)#(mG)#(mA)-TegChol 241 orf1a_6059 (mU)#(mU)#(mU)(mG)(fA)(fU)(fA)(mU)(fU)(mA)(mU)(mC)(mA)#(mC)#(mA)-TegChol 242 orf1a_6322 (mG)#(mA)#(mA)(mU)(fU)(fU)(fG)(mA)(fU)(mG)(mU)(mU)(mU)#(mC)#(mA)-TegChol 243 orf1a_6499 (mC)#(mU)#(mA)(mU)(fU)(fA)(fU)(mU)(fU)(mG)(mC)(mU)(mG)#(mG)#(mA)-TegChol 244 orf1a_7643 (mA)#(mA)#(mA)(mU)(fG)(fU)(fA)(mC)(fU)(mA)(mC)(mC)(mA)#(mG)#(mA)-TegChol 245 orf1a_8200 (mU)#(mC)#(mU)(mU)(fU)(fA)(fG)(mU)(fU)(mU)(mC)(mU)(mA)#(mC)#(mA)-TegChol 246 orf1a_8201 (mA)#(mU)#(mC)(mU)(fU)(fU)(fA)(mG)(fU)(mU)(mU)(mC)(mU)#(mA)#(mA)-TegChol 247 orf1a_8744 (mA)#(mU)#(mG)(mU)(fU)(fU)(fG)(mU)(fU)(mA)(mG)(mC)(mA)#(mA)#(mA)-TegChol 248 orf1a_9679 (mG)#(mC)#(mA)(mA)(fU)(fU)(fG)(mU)(fU)(mA)(mU)(mC)(mC)#(mA)#(mA)-TegChol 249 orf1a_11594 (mU)#(mA)#(mG)(mC)(fA)(fU)(fU)(mA)(fU)(mA)(mC)(mA)(mC)#(mU)#(mA)-TegChol 250 orf1a_12932 (mA)#(mU)#(mA)(mC)(fU)(fU)(fC)(mA)(fC)(mU)(mU)(mU)(mA)#(mG)#(mA)-TegChol 251 S_21944 (mU)#(mU)#(mC)(mA)(fC)(fA)(fG)(mA)(fC)(mU)(mU)(mU)(mA)#(mA)#(mA)-TegChol 252 S_22223 (mU)#(mG)#(mG)(mU)(fU)(fC)(fU)(mA)(fA)(mA)(mG)(mC)(mC)#(mG)#(mA)-TegChol 253 S_22550 (mG)#(mU)#(mU)(mU)(fG)(fU)(fA)(mA)(fU)(mA)(mU)(mU)(mA)#(mG)#(mA)-TegChol 254 S_22820 (mU)#(mU)#(mU)(mA)(fU)(fA)(fA)(mU)(fU)(mA)(mU)(mA)(mA)#(mU)#(mA)-TegChol 255 S_22898 (mA)#(mU)#(mU)(mA)(fU)(fA)(fA)(mU)(fU)(mA)(mC)(mC)(mA)#(mC)#(mA)-TegChol 256 S_23174 (mG)#(mU)#(mU)(mG)(fA)(fA)(fA)(mU)(fU)(mG)(mA)(mC)(mA)#(mC)#(mA)-TegChol 257 S_23239 (mU)#(mG)#(mU)(mU)(fG)(fG)(fA)(mA)(fA)(mG)(mG)(mC)(mA)#(mG)#(mA)-TegChol 258 S_23240 (mU)#(mU)#(mG)(mU)(fU)(fG)(fG)(mA)(fA)(mA)(mG)(mG)(mC)#(mA)#(mA)-TegChol 259 S_23774 (mA)#(mA)#(mU)(mG)(fU)(fA)(fC)(mA)(fU)(mU)(mG)(mU)(mA)#(mC)#(mA)-TegChol 260 S_24056 (mU)#(mU)#(mG)(mU)(fU)(fU)(fG)(mA)(fU)(mG)(mA)(mA)(mG)#(mC)#(mA)-TegChol 261 S_24289 (mU)#(mU)#(mC)(mU)(fG)(fU)(fG)(mU)(fA)(mA)(mC)(mU)(mC)#(mC)#(mA)-TegChol 262 S_25375 (mU)#(mU)#(mC)(mG)(fU)(fU)(fU)(mA)(fU)(mG)(mU)(mG)(mU)#(mA)#(mA)-TegChol 263 3a_25413 (mG)#(mU)#(mU)(mC)(fC)(fA)(fA)(mU)(fU)(mG)(mU)(mG)(mA)#(mA)#(mA)-TegChol 264 3a_25630 (mC)#(mA)#(mG)(mC)(fA)(fA)(fG)(mU)(fU)(mG)(mC)(mA)(mA)#(mA)#(mA)-TegChol 265 3a_25717 (mG)#(mU)#(mA)(mG)(fA)(fC)(fU)(mA)(fA)(mA)(mG)(mC)(mA)#(mU)#(mA)-TegChol 266 3a_25734 (mU)#(mU)#(mU)(mA)(fU)(fA)(fC)(mU)(fC)(mU)(mG)(mC)(mA)#(mA)#(mA)-TegChol 267 3a_25736 (mA)#(mG)#(mU)(mU)(fU)(fA)(fU)(mA)(fC)(mU)(mC)(mU)(mG)#(mC)#(mA)-TegChol 268 3a_25745 (mU)#(mU)#(mC)(mU)(fU)(fA)(fC)(mA)(fA)(mA)(mG)(mU)(mU)#(mU)#(mA)-TegChol 269 3a_25868 (mU)#(mU)#(mA)(mC)(fA)(fC)(fU)(mA)(fU)(mU)(mG)(mU)(mA)#(mA)#(mA)-TegChol 270 3a_25870 (mA)#(mG)#(mU)(mU)(fA)(fC)(fA)(mC)(fU)(mA)(mU)(mU)(mG)#(mU)#(mA)-TegChol 271 3a_25914 (mA)#(mU)#(mA)(mG)(fG)(fA)(fC)(mU)(fU)(mG)(mU)(mU)(mG)#(mU)#(mA)-TegChol 272 3a_25992 (mC)#(mU)#(mG)(mU)(fG)(fU)(fA)(mA)(fU)(mA)(mC)(mA)(mA)#(mC)#(mA)-TegChol 273 3a_26018 (mG)#(mC)#(mU)(mG)(fG)(fU)(fA)(mA)(fU)(mA)(mG)(mU)(mC)#(mU)#(mA)-TegChol 274 3a_26066 (mA)#(mG)#(mG)(mU)(fA)(fA)(fC)(mA)(fU)(mG)(mU)(mU)(mC)#(mA)#(mA)-TegChol 275 E_26258 (mC)#(mU)#(mG)(mU)(fC)(fU)(fC)(mU)(fU)(mC)(mC)(mG)(mA)#(mA)#(mA)-TegChol 276 E_26261 (mU)#(mA)#(mC)(mC)(fU)(fG)(fU)(mC)(fU)(mC)(mU)(mU)(mC)#(mC)#(mA)-TegChol 277 E_26269 (mU)#(mA)#(mU)(mU)(fA)(fA)(fC)(mG)(fU)(mA)(mC)(mC)(mU)#(mG)#(mA)-TegChol 278 E_26277 (mC)#(mU)#(mA)(mU)(fU)(fA)(fA)(mC)(fU)(mA)(mU)(mU)(mA)#(mA)#(mA)-TegChol 279 E_26305 (mU)#(mA)#(mC)(mC)(fA)(fC)(fG)(mA)(fA)(mA)(mG)(mC)(mA)#(mA)#(mA)-TegChol 280 E_26313 (mA)#(mG)#(mC)(mA)(fA)(fG)(fA)(mA)(fU)(mA)(mC)(mC)(mA)#(mC)#(mA)-TegChol 281 E_26369 (mC)#(mA)#(mA)(mU)(fA)(fU)(fU)(mG)(fC)(mA)(mG)(mC)(mA)#(mG)#(mA)-TegChol 282 E_26374 (mG)#(mU)#(mU)(mA)(fA)(fC)(fA)(mA)(fU)(mA)(mU)(mU)(mG)#(mC)#(mA)-TegChol 283 E_26455 (mG)#(mA)#(mC)(mC)(fA)(fG)(fA)(mA)(fG)(mA)(mU)(mC)(mA)#(mG)#(mA)-TegChol 284 E_26463 (mU)#(mU)#(mC)(mG)(fU)(fU)(fU)(mA)(fG)(mA)(mC)(mC)(mA)#(mG)#(mA)-TegChol 285 E_26467 (mU)#(mU)#(mA)(mG)(fU)(fU)(fC)(mG)(fU)(mU)(mU)(mA)(mG)#(mA)#(mA)-TegChol 286 E_26470 (mU)#(mA)#(mU)(mU)(fU)(fA)(fG)(mU)(fU)(mC)(mG)(mU)(mU)#(mU)#(mA)-TegChol 287 M_26573 (mA)#(mG)#(mG)(mU)(fU)(fC)(fC)(mA)(fU)(mU)(mG)(mU)(mU)#(mC)#(mA)-TegChol 288 M_26581 (mC)#(mU)#(mA)(mU)(fU)(fA)(fC)(mU)(fA)(mG)(mG)(mU)(mU)#(mC)#(mA)-TegChol 289 M_26602 (mU)#(mC)#(mC)(mA)(fU)(fG)(fU)(mA)(fA)(mG)(mG)(mA)(mA)#(mU)#(mA)-TegChol 290 M_26624 (mU)#(mA)#(mG)(mG)(fC)(fA)(fA)(mA)(fU)(mU)(mG)(mU)(mA)#(mG)#(mA)-TegChol 291 M_26637 (mA)#(mU)#(mU)(mC)(fC)(fU)(fG)(mU)(fU)(mG)(mG)(mC)(mA)#(mU)#(mA)-TegChol 292 M_26638 (mU)#(mA)#(mU)(mU)(fC)(fC)(fU)(mG)(fU)(mU)(mG)(mG)(mC)#(mA)#(mA)-TegChol 293 M_26693 (mA)#(mA)#(mA)(mG)(fU)(fU)(fA)(mC)(fU)(mG)(mG)(mC)(mC)#(mA)#(mA)-TegChol 294 M_26717 (mA)#(mC)#(mA)(mG)(fC)(fA)(fG)(mC)(fA)(mA)(mG)(mC)(mA)#(mC)#(mA)-TegChol 295 M_27014 (mG)#(mU)#(mG)(mA)(fU)(fU)(fU)(mC)(fU)(mU)(mU)(mA)(mG)#(mG)#(mA)-TegChol 296 M_27032 (mG)#(mU)#(mU)(mC)(fG)(fU)(fG)(mA)(fU)(mG)(mU)(mA)(mG)#(mC)#(mA)-TegChol 297 M_27035 (mA)#(mG)#(mC)(mG)(fU)(fU)(fC)(mG)(fU)(mG)(mA)(mU)(mG)#(mU)#(mA)-TegChol 298 M_27123 (mU)#(mU)#(mU)(mA)(fU)(fA)(fG)(mU)(fU)(mG)(mC)(mC)(mA)#(mA)#(mA)-TegChol 299 7a_27455 (mC)#(mU)#(mC)(mU)(fA)(fA)(fC)(mA)(fC)(mA)(mC)(mU)(mC)#(mU)#(mA)-TegChol 300 7a_27522 (mG)#(mG)#(mA)(mU)(fG)(fA)(fA)(mA)(fU)(mG)(mG)(mU)(mG)#(mA)#(mA)-TegChol 301 7a_27537 (mU)#(mU)#(mG)(mU)(fU)(fA)(fU)(mC)(fA)(mG)(mC)(mU)(mA)#(mG)#(mA)-TegChol 302 7a_27553 (mG)#(mC)#(mA)(mA)(fG)(fU)(fC)(mA)(fG)(mU)(mG)(mC)(mA)#(mA)#(mA)-TegChol 303 7a_27565 (mU)#(mU)#(mG)(mA)(fG)(fU)(fG)(mC)(fU)(mA)(mA)(mA)(mG)#(mC)#(mA)-TegChol 304 7a_27633 (mU)#(mU)#(mA)(mG)(fG)(fU)(fG)(mA)(fA)(mA)(mC)(mU)(mG)#(mA)#(mA)-TegChol 305 7a_27656 (mC)#(mU)#(mU)(mC)(fC)(fU)(fC)(mU)(fU)(mG)(mU)(mC)(mU)#(mG)#(mA)-TegChol 306 7a_27671 (mA)#(mG)#(mU)(mA)(fA)(fA)(fG)(mU)(fU)(mC)(mU)(mU)(mG)#(mA)#(mA)-TegChol 307 7a_27705 (mA)#(mA)#(mC)(mA)(fC)(fU)(fA)(mU)(fU)(mG)(mC)(mC)(mG)#(mC)#(mA)-TegChol 308 7a_27715 (mA)#(mA)#(mG)(mU)(fG)(fU)(fU)(mA)(fU)(mA)(mA)(mA)(mC)#(mA)#(mA)-TegChol 309 7a_27720 (mA)#(mA)#(mG)(mC)(fA)(fA)(fA)(mG)(fU)(mG)(mU)(mU)(mA)#(mU)#(mA)-TegChol 310 7a_27751 (mG)#(mU)#(mU)(mC)(fA)(fA)(fU)(mC)(fA)(mU)(mU)(mC)(mU)#(mG)#(mA)-TegChol 311 8b_27932 (mU)#(mG)#(mG)(mU)(fG)(fA)(fA)(mA)(fU)(mG)(mC)(mA)(mG)#(mC)#(mA)-TegChol 312 8b_27940 (mU)#(mA)#(mC)(mA)(fU)(fU)(fC)(mU)(fU)(mG)(mG)(mU)(mG)#(mA)#(mA)-TegChol 313 8b_27986 (mG)#(mG)#(mG)(mU)(fC)(fA)(fU)(mC)(fA)(mA)(mC)(mU)(mA)#(mC)#(mA)-TegChol 314 8b_28002 (mG)#(mA)#(mA)(mG)(fU)(fG)(fA)(mA)(fU)(mA)(mG)(mG)(mA)#(mC)#(mA)-TegChol 315 8b_28024 (mC)#(mU)#(mC)(mU)(fA)(fA)(fU)(mA)(fU)(mA)(mC)(mC)(mA)#(mU)#(mA)-TegChol 316 8b_28091 (mA)#(mU)#(mG)(mG)(fG)(fU)(fG)(mA)(fU)(mU)(mU)(mA)(mG)#(mA)#(mA)-TegChol 317 orf1ab_15376 (mA)#(mC)#(mG)(mG)(mU)(mG)(mU)(mG)(mA)(mC)(mA)(mA)(mG)(mC)(mU)(mA)#(mC)#(mA) 339 orf1ab_15786 (mU)#(mA)#(mA)(mU)(mA)(mA)(mA)(mG)(mA)(mA)(mC)(mU)(mG)(mA)(mC)(mU)#(mU)#(mA) 340 orf1ab_17107 (mA)#(mG)#(mC)(mU)(mA)(mG)(mG)(mC)(mC)(mA)(mA)(mU)(mA)(mG)(mC)(mA)#(mA)#(mA) 341 orf1ab_17370 (mA)#(mA)#(mA)(mU)(mC)(mA)(mU)(mA)(mA)(mU)(mU)(mU)(mG)(mU)(mG)(mG)#(mC)#(mA) 342 orf1ab_18025 (mA)#(mG)#(mC)(mU)(mU)(mG)(mU)(mA)(mA)(mA)(mG)(mU)(mU)(mG)(mC)(mC)#(mA)#(mA) 343 orf1ab_18571 (mA)#(mA)#(mA)(mU)(mA)(mC)(mG)(mA)(mC)(mU)(mC)(mU)(mG)(mU)(mC)(mA)#(mG)#(mA) 344 orf1ab_20497 (mU)#(mA)#(mA)(mU)(mA)(mA)(mA)(mU)(mC)(mA)(mA)(mU)(mA)(mA)(mC)(mA)#(mG)#(mA) 345 orf1ab_20892 (mA)#(mC)#(mA)(mG)(mC)(mU)(mG)(mU)(mA)(mC)(mC)(mU)(mG)(mG)(mU)(mG)#(mC)#(mA) 346 orf1ab_21391 (mA)#(mA)#(mA)(mU)(mU)(mU)(mA)(mC)(mU)(mC)(mA)(mU)(mG)(mU)(mC)(mA)#(mA)#(mA) 347 orf1a_416 (mA)#(mA)#(mC)(mU)(mU)(mC)(mU)(mA)(mC)(mU)(mA)(mA)(mG)(mC)(mC)(mA)#(mC)#(mA) 348 orf1a_2290 (mA)#(mG)#(mC)(mU)(mU)(mA)(mA)(mA)(mG)(mA)(mA)(mU)(mG)(mU)(mC)(mU)#(mG)#(mA) 349 orf1a_6059 (mA)#(mA)#(mA)(mU)(mU)(mU)(mG)(mA)(mU)(mA)(mU)(mU)(mA)(mU)(mC)(mA)#(mC)#(mA) 350 orf1a_6322 (mA)#(mA)#(mC)(mG)(mA)(mA)(mU)(mU)(mU)(mG)(mA)(mU)(mG)(mU)(mU)(mU)#(mC)#(mA) 351 orf1a_6499 (mA)#(mA)#(mA)(mC)(mU)(mA)(mU)(mU)(mA)(mU)(mU)(mU)(mG)(mC)(mU)(mG)#(mG)#(mA) 352 orf1a_7643 (mA)#(mA)#(mU)(mA)(mA)(mA)(mU)(mG)(mU)(mA)(mC)(mU)(mA)(mC)(mC)(mA)#(mG)#(mA) 353 orf1a_8200 (mA)#(mC)#(mA)(mU)(mC)(mU)(mU)(mU)(mA)(mG)(mU)(mU)(mU)(mC)(mU)(mA)#(mC)#(mA) 354 orf1a_8201 (mA)#(mA)#(mC)(mA)(mU)(mC)(mU)(mU)(mU)(mA)(mG)(mU)(mU)(mU)(mC)(mU)#(mA)#(mA) 355 orf1a_8744 (mA)#(mG)#(mC)(mA)(mU)(mG)(mU)(mU)(mU)(mG)(mU)(mU)(mA)(mG)(mC)(mA)#(mA)#(mA) 356 orf1a_9679 (mU)#(mA)#(mA)(mG)(mC)(mA)(mA)(mU)(mU)(mG)(mU)(mU)(mA)(mU)(mC)(mC)#(mA)#(mA) 357 orf1a_11594 (mA)#(mA)#(mC)(mU)(mA)(mG)(mC)(mA)(mU)(mU)(mA)(mU)(mA)(mC)(mA)(mC)#(mU)#(mA) 358 orf1a_12932 (mU)#(mA)#(mA)(mA)(mU)(mA)(mC)(mU)(mU)(mC)(mA)(mC)(mU)(mU)(mU)(mA)#(mG)#(mA) 359 S_21944 (mA)#(mA)#(mA)(mU)(mU)(mC)(mA)(mC)(mA)(mG)(mA)(mC)(mU)(mU)(mU)(mA)#(mA)#(mA) 360 S_22223 (mC)#(mA)#(mA)(mU)(mG)(mG)(mU)(mU)(mC)(mU)(mA)(mA)(mA)(mG)(mC)(mC)#(mG)#(mA) 361 S_22550 (mC)#(mA)#(mA)(mG)(mU)(mU)(mU)(mG)(mU)(mA)(mA)(mU)(mA)(mU)(mU)(mA)#(mG)#(mA) 362 S_22820 (mU)#(mA)#(mA)(mU)(mU)(mU)(mA)(mU)(mA)(mA)(mU)(mU)(mA)(mU)(mA)(mA)#(mU)#(mA) 363 S_22898 (mG)#(mU)#(mA)(mA)(mU)(mU)(mA)(mU)(mA)(mA)(mU)(mU)(mA)(mC)(mC)(mA)#(mC)#(mA) 364 S_23174 (mG)#(mA)#(mA)(mG)(mU)(mU)(mG)(mA)(mA)(mA)(mU)(mU)(mG)(mA)(mC)(mA)#(mC)#(mA) 365 S_23239 (mA)#(mA)#(mU)(mU)(mG)(mU)(mU)(mG)(mG)(mA)(mA)(mA)(mG)(mG)(mC)(mA)#(mG)#(mA) 366 S_23240 (mA)#(mA)#(mA)(mU)(mU)(mG)(mU)(mU)(mG)(mG)(mA)(mA)(mA)(mG)(mG)(mC)#(mA)#(mA) 367 S_23774 (mA)#(mC)#(mA)(mA)(mA)(mU)(mG)(mU)(mA)(mC)(mA)(mU)(mU)(mG)(mU)(mA)#(mC)#(mA) 368 S_24056 (mA)#(mU)#(mA)(mU)(mU)(mG)(mU)(mU)(mU)(mG)(mA)(mU)(mG)(mA)(mA)(mG)#(mC)#(mA) 369 S_24289 (mA)#(mC)#(mA)(mU)(mU)(mC)(mU)(mG)(mU)(mG)(mU)(mA)(mA)(mC)(mU)(mC)#(mC)#(mA) 370 S_25375 (mA)#(mA)#(mG)(mU)(mU)(mC)(mG)(mU)(mU)(mU)(mA)(mU)(mG)(mU)(mG)(mU)#(mA)#(mA) 371 3a_25413 (mA)#(mC)#(mA)(mG)(mU)(mU)(mC)(mC)(mA)(mA)(mU)(mU)(mG)(mU)(mG)(mA)#(mA)#(mA) 372 3a_25630 (mC)#(mA)#(mA)(mC)(mA)(mG)(mC)(mA)(mA)(mG)(mU)(mU)(mG)(mC)(mA)(mA)#(mA)#(mA) 373 3a_25717 (mG)#(mA)#(mA)(mG)(mU)(mA)(mG)(mA)(mC)(mU)(mA)(mA)(mA)(mG)(mC)(mA)#(mU)#(mA) 374 3a_25734 (mA)#(mA)#(mG)(mU)(mU)(mU)(mA)(mU)(mA)(mC)(mU)(mC)(mU)(mG)(mC)(mA)#(mA)#(mA) 375 3a_25736 (mC)#(mA)#(mA)(mA)(mG)(mU)(mU)(mU)(mA)(mU)(mA)(mC)(mU)(mC)(mU)(mG)#(mC)#(mA) 376 3a_25745 (mU)#(mU)#(mA)(mU)(mU)(mC)(mU)(mU)(mA)(mC)(mA)(mA)(mA)(mG)(mU)(mU)#(mU)#(mA) 377 3a_25868 (mA)#(mA)#(mG)(mU)(mU)(mA)(mC)(mA)(mC)(mU)(mA)(mU)(mU)(mG)(mU)(mA)#(mA)#(mA) 378 3a_25870 (mA)#(mG)#(mA)(mA)(mG)(mU)(mU)(mA)(mC)(mA)(mC)(mU)(mA)(mU)(mU)(mG)#(mU)#(mA) 379 3a_25914 (mG)#(mA)#(mA)(mA)(mU)(mA)(mG)(mG)(mA)(mC)(mU)(mU)(mG)(mU)(mU)(mG)#(mU)#(mA) 380 3a_25992 (mU)#(mA)#(mA)(mC)(mU)(mG)(mU)(mG)(mU)(mA)(mA)(mU)(mA)(mC)(mA)(mA)#(mC)#(mA) 381 3a_26018 (mA)#(mC)#(mA)(mG)(mC)(mU)(mG)(mG)(mU)(mA)(mA)(mU)(mA)(mG)(mU)(mC)#(mU)#(mA) 382 3a_26066 (mA)#(mG)#(mA)(mA)(mG)(mG)(mU)(mA)(mA)(mC)(mA)(mU)(mG)(mU)(mU)(mC)#(mA)#(mA) 383 E_26258 (mU)#(mA)#(mC)(mC)(mU)(mG)(mU)(mC)(mU)(mC)(mU)(mU)(mC)(mC)(mG)(mA)#(mA)#(mA) 384 E_26261 (mA)#(mC)#(mG)(mU)(mA)(mC)(mC)(mU)(mG)(mU)(mC)(mU)(mC)(mU)(mU)(mC)#(mC)#(mA) 385 E_26269 (mA)#(mA)#(mC)(mU)(mA)(mU)(mU)(mA)(mA)(mC)(mG)(mU)(mA)(mC)(mC)(mU)#(mG)#(mA) 386 E_26277 (mA)#(mC)#(mG)(mC)(mU)(mA)(mU)(mU)(mA)(mA)(mC)(mU)(mA)(mU)(mU)(mA)#(mA)#(mA) 387 E_26305 (mG)#(mA)#(mA)(mU)(mA)(mC)(mC)(mA)(mC)(mG)(mA)(mA)(mA)(mG)(mC)(mA)#(mA)#(mA) 388 E_26313 (mA)#(mC)#(mU)(mA)(mG)(mC)(mA)(mA)(mG)(mA)(mA)(mU)(mA)(mC)(mC)(mA)#(mC)#(mA) 389 E_26369 (mU)#(mA)#(mA)(mC)(mA)(mA)(mU)(mA)(mU)(mU)(mG)(mC)(mA)(mG)(mC)(mA)#(mG)#(mA) 390 E_26374 (mC)#(mA)#(mC)(mG)(mU)(mU)(mA)(mA)(mC)(mA)(mA)(mU)(mA)(mU)(mU)(mG)#(mC)#(mA) 391 E_26455 (mU)#(mU)#(mA)(mG)(mA)(mC)(mC)(mA)(mG)(mA)(mA)(mG)(mA)(mU)(mC)(mA)#(mG)#(mA) 392 E_26463 (mU)#(mA)#(mG)(mU)(mU)(mC)(mG)(mU)(mU)(mU)(mA)(mG)(mA)(mC)(mC)(mA)#(mG)#(mA) 393 E_26467 (mU)#(mA)#(mU)(mU)(mU)(mA)(mG)(mU)(mU)(mC)(mG)(mU)(mU)(mU)(mA)(mG)#(mA)#(mA) 394 E_26470 (mU)#(mA)#(mA)(mU)(mA)(mU)(mU)(mU)(mA)(mG)(mU)(mU)(mC)(mG)(mU)(mU)#(mU)#(mA) 395 M_26573 (mA)#(mC)#(mU)(mA)(mG)(mG)(mU)(mU)(mC)(mC)(mA)(mU)(mU)(mG)(mU)(mU)#(mC)#(mA) 396 M_26581 (mA)#(mA)#(mC)(mC)(mU)(mA)(mU)(mU)(mA)(mC)(mU)(mA)(mG)(mG)(mU)(mU)#(mC)#(mA) 397 M_26602 (mA)#(mA)#(mA)(mU)(mC)(mC)(mA)(mU)(mG)(mU)(mA)(mA)(mG)(mG)(mA)(mA)#(mU)#(mA) 398 M_26624 (mG)#(mC)#(mA)(mU)(mA)(mG)(mG)(mC)(mA)(mA)(mA)(mU)(mU)(mG)(mU)(mA)#(mG)#(mA) 399 M_26637 (mC)#(mC)#(mU)(mA)(mU)(mU)(mC)(mC)(mU)(mG)(mU)(mU)(mG)(mG)(mC)(mA)#(mU)#(mA) 400 M_26638 (mA)#(mC)#(mC)(mU)(mA)(mU)(mU)(mC)(mC)(mU)(mG)(mU)(mU)(mG)(mG)(mC)#(mA)#(mA) 401 M_26693 (mG)#(mC)#(mU)(mA)(mA)(mA)(mG)(mU)(mU)(mA)(mC)(mU)(mG)(mG)(mC)(mC)#(mA)#(mA) 402 M_26717 (mU)#(mA)#(mA)(mA)(mC)(mA)(mG)(mC)(mA)(mG)(mC)(mA)(mA)(mG)(mC)(mA)#(mC)#(mA) 403 M_27014 (mA)#(mC)#(mA)(mG)(mU)(mG)(mA)(mU)(mU)(mU)(mC)(mU)(mU)(mU)(mA)(mG)#(mG)#(mA) 404 M_27032 (mA)#(mG)#(mC)(mG)(mU)(mU)(mC)(mG)(mU)(mG)(mA)(mU)(mG)(mU)(mA)(mG)#(mC)#(mA) 405 M_27035 (mG)#(mA)#(mA)(mA)(mG)(mC)(mG)(mU)(mU)(mC)(mG)(mU)(mG)(mA)(mU)(mG)#(mU)#(mA) 406 M_27123 (mU)#(mA)#(mA)(mU)(mU)(mU)(mA)(mU)(mA)(mG)(mU)(mU)(mG)(mC)(mC)(mA)#(mA)#(mA) 407 7a_27455 (mU)#(mA)#(mC)(mC)(mU)(mC)(mU)(mA)(mA)(mC)(mA)(mC)(mA)(mC)(mU)(mC)#(mU)#(mA) 408 7a_27522 (mA)#(mG)#(mA)(mG)(mG)(mA)(mU)(mG)(mA)(mA)(mA)(mU)(mG)(mG)(mU)(mG)#(mA)#(mA) 409 7a_27537 (mA)#(mA)#(mU)(mU)(mU)(mG)(mU)(mU)(mA)(mU)(mC)(mA)(mG)(mC)(mU)(mA)#(mG)#(mA) 410 7a_27553 (mA)#(mA)#(mA)(mG)(mC)(mA)(mA)(mG)(mU)(mC)(mA)(mG)(mU)(mG)(mC)(mA)#(mA)#(mA) 411 7a_27565 (mA)#(mA)#(mA)(mU)(mU)(mG)(mA)(mG)(mU)(mG)(mC)(mU)(mA)(mA)(mA)(mG)#(mC)#(mA) 412 7a_27633 (mA)#(mG)#(mU)(mU)(mU)(mA)(mG)(mG)(mU)(mG)(mA)(mA)(mA)(mC)(mU)(mG)#(mA)#(mA) 413 7a_27656 (mG)#(mA)#(mA)(mC)(mU)(mU)(mC)(mC)(mU)(mC)(mU)(mU)(mG)(mU)(mC)(mU)#(mG)#(mA) 414 7a_27671 (mG)#(mA)#(mG)(mA)(mG)(mU)(mA)(mA)(mA)(mG)(mU)(mU)(mC)(mU)(mU)(mG)#(mA)#(mA) 415 7a_27705 (mA)#(mU)#(mA)(mA)(mA)(mC)(mA)(mC)(mU)(mA)(mU)(mU)(mG)(mC)(mC)(mG)#(mC)#(mA) 416 7a_27715 (mG)#(mC)#(mA)(mA)(mA)(mG)(mU)(mG)(mU)(mU)(mA)(mU)(mA)(mA)(mA)(mC)#(mA)#(mA) 417 7a_27720 (mG)#(mU)#(mG)(mA)(mA)(mG)(mC)(mA)(mA)(mA)(mG)(mU)(mG)(mU)(mU)(mA)#(mU)#(mA) 418 7a_27751 (mA)#(mA)#(mA)(mG)(mU)(mU)(mC)(mA)(mA)(mU)(mC)(mA)(mU)(mU)(mC)(mU)#(mG)#(mA) 419 8b_27932 (mU)#(mC)#(mU)(mU)(mG)(mG)(mU)(mG)(mA)(mA)(mA)(mU)(mG)(mC)(mA)(mG)#(mC)#(mA) 420 8b_27940 (mA)#(mA)#(mC)(mU)(mA)(mC)(mA)(mU)(mU)(mC)(mU)(mU)(mG)(mG)(mU)(mG)#(mA)#(mA) 421 8b_27986 (mC)#(mA)#(mC)(mG)(mG)(mG)(mU)(mC)(mA)(mU)(mC)(mA)(mA)(mC)(mU)(mA)#(mC)#(mA) 422 8b_28002 (mA)#(mU)#(mA)(mG)(mA)(mA)(mG)(mU)(mG)(mA)(mA)(mU)(mA)(mG)(mG)(mA)#(mC)#(mA) 423 8b_28024 (mC)#(mU)#(mA)(mC)(mU)(mC)(mU)(mA)(mA)(mU)(mA)(mU)(mA)(mC)(mC)(mA)#(mU)#(mA) 424 8b_28091 (mU)#(mG)#(mA)(mA)(mU)(mG)(mG)(mG)(mU)(mG)(mA)(mU)(mU)(mU)(mA)(mG)#(mA)#(mA) 425 8b_28119 (mA)#(mA)#(mC)(mU)(mG)(mU)(mA)(mU)(mA)(mA)(mU)(mU)(mA)(mC)(mC)(mG)#(mA)#(mA) 426 8b_28127 (mA)#(mA)#(mA)(mC)(mA)(mG)(mG)(mA)(mA)(mA)(mC)(mU)(mG)(mU)(mA)(mU)#(mA)#(mA) 427 8b_28128 (mU)#(mA)#(mA)(mA)(mC)(mA)(mG)(mG)(mA)(mA)(mA)(mC)(mU)(mG)(mU)(mA)#(mU)#(mA) 428 8b_28163 (mC)#(mC)#(mC)(mA)(mA)(mU)(mU)(mU)(mA)(mG)(mG)(mU)(mU)(mC)(mC)(mU)#(mG)#(mA) 429 8b_28218 (mA)#(mA)#(mC)(mG)(mU)(mC)(mA)(mU)(mG)(mA)(mU)(mA)(mC)(mU)(mC)(mU)#(mA)#(mA) 430 8b_28222 (mC)#(mA)#(mC)(mG)(mA)(mA)(mC)(mG)(mU)(mC)(mA)(mU)(mG)(mA)(mU)(mA)#(mC)#(mA) 431 N_28407 (mA)#(mC)#(mG)(mC)(mA)(mG)(mU)(mA)(mU)(mU)(mA)(mU)(mU)(mG)(mG)(mG)#(mU)#(mA) 432 N_28655 (mA)#(mA)#(mC)(mC)(mC)(mA)(mU)(mA)(mU)(mG)(mA)(mU)(mG)(mC)(mC)(mG)#(mU)#(mA) 433 N_28945 (mA)#(mG)#(mC)(mU)(mG)(mG)(mU)(mU)(mC)(mA)(mA)(mU)(mC)(mU)(mG)(mU)#(mC)#(mA) 434 N_28992 (mC)#(mA)#(mG)(mU)(mU)(mU)(mG)(mG)(mC)(mC)(mU)(mU)(mG)(mU)(mU)(mG)#(mU)#(mA) 435 N_29141 (mU)#(mC)#(mC)(mU)(mU)(mG)(mU)(mC)(mU)(mG)(mA)(mU)(mU)(mA)(mG)(mU)#(mU)#(mA) 436 N_29276 (mA)#(mU)#(mC)(mC)(mA)(mA)(mU)(mU)(mU)(mG)(mA)(mU)(mG)(mG)(mC)(mA)#(mC)#(mA) 437 N_29292 (mA)#(mA)#(mU)(mU)(mU)(mG)(mG)(mA)(mU)(mC)(mU)(mU)(mU)(mG)(mU)(mC)#(mA)#(mA) 438 N_29293 (mA)#(mA)#(mA)(mU)(mU)(mU)(mG)(mG)(mA)(mU)(mC)(mU)(mU)(mU)(mG)(mU)#(mC)#(mA) 439 N_29303 (mU)#(mU)#(mG)(mA)(mU)(mC)(mU)(mU)(mU)(mG)(mA)(mA)(mA)(mU)(mU)(mU)#(mG)#(mA) 440 N_29307 (mU)#(mG)#(mA)(mC)(mU)(mU)(mG)(mA)(mU)(mC)(mU)(mU)(mU)(mG)(mA)(mA)#(mA)#(mA) 441 N_29328 (mC)#(mA)#(mA)(mU)(mA)(mU)(mG)(mC)(mU)(mU)(mA)(mU)(mU)(mC)(mA)(mG)#(mC)#(mA) 442 N_29464 (mA)#(mA)#(mA)(mU)(mC)(mA)(mU)(mC)(mC)(mA)(mA)(mA)(mU)(mC)(mU)(mG)#(mC)#(mA) 443

TABLE 7 SARS-CoV2-Additional target sequences SEQ SEQ ID ID Sequence ID  20 nt Sequence NO: 45 nt Gene Region NO: 3a_25688 CUGGCCUUGAAGCC 444 TTTTGCTCGTTGCTGCT 642 CCUUUU GGCCTTGAAGCCCCTT TTCTCTATCTTT 3a_25570 UCCAAAAUCAUAAC 445 GTTTTTCAGAGCGCTTC 643 CCUCAA CAAAATCATAACCCTC AAAAAGAGATGG 3a_25852 UACGACUAUUGUA 446 TGGCATACTAATTGTT 644 UACCUUA ACGACTATTGTATACC TTACAATAGTGTA 3a_25518 UUUCGGAUGGCUU 447 ACAAGCCTCACTCCCT 645 AUUGUUG TTCGGATGGCTTATTGT TGGCGTTGCACT 3a_25974 AUCUGGAGUAAAA 448 TACTGAAAAATGGGAA 646 GACUGUG TCTGGAGTAAAAGACT GTGTTGTATTACA 3a_25835 GCUGGCAUACUAA 449 CCAACTATTTTCTTTGC 647 UUGUUAC TGGCATACTAATTGTT ACGACTATTGTA 3a_25645 UUGUUGUUUGUAA 450 GTTTGCAACTTGCTGTT 648 CAGUUUA GTTGTTTGTAACAGTTT ACTCACACCTT 3a_25773 UUGGCUUUGCUGG 451 AATAATAATGAGGCTT 649 AAAUGCC TGGCTTTGCTGGAAAT GCCGTTCCAAAAA 3a_25921 AGUCCUAUUUCUG 452 GGTGATGGCACAACAA 650 AACAUGA GTCCTATTTCTGAACAT GACTACCAGATT 3a_26114 AAGAACAUGUCCA 453 TTGTTGATGAGCCTGA 651 AAUUCAC AGAACATGTCCAAATT CACACAATCGACG 3a_25750 UUUGUAAGAAUAA 454 TTGCAGAGTATAAACT 652 UAAUGAG TTGTAAGAATAATAAT GAGGCTTTGGCTT 3a_25862 GUAUACCUUACAA 455 ATTGTTACGACTATTGT 653 UAGUGUA ATACCTTACAATAGTG TAACTTCTTCAA 3a_25847 AUUGUUACGACUA 456 TTTGCTGGCATACTAA 654 UUGUAUA TTGTTACGACTATTGTA TACCTTACAATA 3a_26105 AUGAGCCUGAAGA 457 ACAATAAAATTGTTGA 655 ACAUGUC TGAGCCTGAAGAACAT GTCCAAATTCACA 3a_25863 UAUACCUUACAAU 458 TTGTTACGACTATTGTA 656 AGUGUAA TACCTTACAATAGTGT AACTTCTTCAAT 3a_26164 CCAGUAAUGGAACC 459 TCCGGAGTTGTTAATC 657 AAUUUA CAGTAATGGAACCAAT TTATGATGAACCG 3a_25706 UUCUCUAUCUUUA 460 GCCTTGAAGCCCCTTTT  658 UGCUUUA CTCTATCTTTATGCTTT AGTCTACTTCT 3a_25595 GAUGGCAACUAGC 461 TAACCCTCAAAAAGAG 659 ACUCUCC ATGGCAACTAGCACTC TCCAAGGGTGTTC 3a_25611 CUCCAAGGGUGUUC 462 ATGGCAACTAGCACTC 660 ACUUUG TCCAAGGGTGTTCACT TTGTTTGCAACTT 3a_25405 AUGAGAAUCUUCA 463 CTTATGGATTTGTTTAT 661 CAAUUGG GAGAATCTTCACAATT GGAACTGTAACT 3a_25535 UUGGCGUUGCACU 464 TCGGATGGCTTATTGTT 662 UCUUGCU GGCGTTGCACTTCTTG CTGTTTTTCAGA 3a_25653 UGUAACAGUUUAC 465 CTTGCTGTTGTTGTTTG 663 UCACACC TAACAGTTTACTCACA CCTTTTGCTCGT 7a_27598 GACGGCGUAAAAC 466 GCTTTTGCTTGTCCTGA 664 ACGUCUA CGGCGTAAAACACGTC TATCAGTTACGT 7a_27483 UUUAAAAGAACCU 467 AGGTACAACAGTACTT 665 UGCUCUU TTAAAAGAACCTTGCT CTTCTGGAACATA 7a_27258 GAGGACUUUUAAA 468 ATTACTAATTATTATG 666 GUUUCCA AGGACTTTTAAAGTTT CCATTTGGAATCT 7a_27264 UUUUAAAGUUUCC 469 AATTATTATGAGGACT 667 AUUUGGA TTTAAAGTTTCCATTTG GAATCTTGATTA 7a_27257 UGAGGACUUUUAA 470 TATTACTAATTATTATG 668 AGUUUCC AGGACTTTTAAAGTTT CCATTTGGAATC 7a_27334 ACUGAGAAUAAAU 471 TTATCTAAGTCACTAA 669 AUUCUCA CTGAGAATAAATATTC TCAATTAGATGAA 7a_27595 CCUGACGGCGUAAA 472 TTTGCTTTTGCTTGTCC 670 ACACGU TGACGGCGTAAAACAC GTCTATCAGTTA 7a_27466 AGAGGUACAACAG 473 TACCAAGAGTGTGTTA 671 UACUUUU GAGGTACAACAGTACT TTTAAAAGAACCT 7a_27237 AGAGAUAUUACUA 474 TCAGGTTACTATAGCA 672 AUUAUUA GAGATATTACTAATTA TTATGAGGACTTT 7a_27395 UGAAAAUUAUUCU 475 TTGATTAAACGAACAT 673 UUUCUUG GAAAATTATTCTTTTCT TGGCACTGATAA 7a_27289 GAUUACAUCAUAA 476 TCCATTTGGAATCTTG 674 ACCUCAU ATTACATCATAAACCT CATAATTAAAAAT 7a_27243 AUUACUAAUUAUU 477 TACTATAGCAGAGATA 675 AUGAGGA TTACTAATTATTATGA GGACTTTTAAAGT 7a_27319 UUAUCUAAGUCAC 478 CTCATAATTAAAAATT 676 UAACUGA TATCTAAGTCACTAAC TGAGAATAAATAT 7a_27226 GUUACUAUAGCAG 479 CTCGTTGACTTTCAGGT 677 AGAUAUU TACTATAGCAGAGATA TTACTAATTATT 7a_27256 AUGAGGACUUUUA 480 ATATTACTAATTATTAT 678 AAGUUUC GAGGACTTTTAAAGTT TCCATTTGGAAT 7a_27203 UGUUUCAUCUCGU 481 GTAAGTGACAACAGAT 679 UGACUUU GTTTCATCTCGTTGACT TTCAGGTTACTA 7a_27292 UACAUCAUAAACCU 482 ATTTGGAATCTTGATT 680 CAUAAU ACATCATAAACCTCAT AATTAAAAATTTA 7a_27333 AACUGAGAAUAAA 483 TTTATCTAAGTCACTA 681 UAUUCUC ACTGAGAATAAATATT CTCAATTAGATGA 7a_27255 UAUGAGGACUUUU 484 GATATTACTAATTATT 682 AAAGUUU ATGAGGACTTTTAAAG TTTCCATTTGGAA 7a_27381 UGAUUAAACGAAC 485 GCAACCAATGGAGATT 683 AUGAAAA GATTAAACGAACATGA AAATTATTCTTTT 7a_27750 GACAGAAUGAUUG 486 CACACTCAAAAGAAAG 684 AACUUUC ACAGAATGATTGAACT TTCATTAATTGAC 7a_27445 UAUCACUACCAAGA 487 GCTACTTGTGAGCTTT 685 GUGUGU ATCACTACCAAGAGTG TGTTAGAGGTACA 8b_27795 UUAGCCUUUCUGCU 488 TTCTATTTGTGCTTTTT 686 AUUCCU AGCCTTTCTGCTATTCC TTGTTTTAATT 8b_27803 UCUGCUAUUCCUUG 489 GTGCTTTTTAGCCTTTC 687 UUUUAA TGCTATTCCTTGTTTTA ATTATGCTTAT 8b_27888 ACGAACAUGAAAU 490 ACTTGTCACGCCTAAA 688 UUCUUGU CGAACATGAAATTTCT TGTTTTCTTAGGA 8b_28236 CGUGUUGUUUUAG 491 GAGTATCATGACGTTC 689 AUUUCAU GTGTTGTTTTAGATTTC ATCTAAACGAAC 8b_27895 UGAAAUUUCUUGU 492 ACGCCTAAACGAACAT 690 UUUCUUA GAAATTTCTTGTTTTCT TAGGAATCATCA 8b_28142 UUUACCUUUUACA 493 TTATACAGTTTCCTGTT 691 AUUAAUU TACCTTTTACAATTAAT TGCCAGGAACC 8b_27802 UUCUGCUAUUCCUU 494 TGTGCTTTTTAGCCTTT 692 GUUUUA CTGCTATTCCTTGTTTT AATTATGCTTA 8b_27808 UAUUCCUUGUUUU 495 TTTTAGCCTTTCTGCTA 693 AAUUAUG TTCCTTGTTTTAATTAT GCTTATTATCT 8b_27796 UAGCCUUUCUGCUA 496 TCTATTTGTGCTTTTTA 694 UUCCUU GCCTTTCTGCTATTCCT TGTTTTAATTA 8b_27815 UGUUUUAAUUAUG 497 CTTTCTGCTATTCCTTG 695 CUUAUUA TTTTAATTATGCTTATT ATCTTTTGGTT 8b_28044 GCUAGAAAAUCAG 498 TATATTAGAGTAGGAG 696 CACCUUU CTAGAAAATCAGCACC TTTAATTGAATTG 8b_27794 UUUAGCCUUUCUGC 499 CTTCTATTTGTGCTTTT 697 UAUUCC TAGCCTTTCTGCTATTC CTTGTTTTAAT 8b_28234 UUCGUGUUGUUUU 500 TAGAGTATCATGACGT 698 AGAUUUC TCGTGTTGTTTTAGATT TCATCTAAACGA 8b_27817 UUUUAAUUAUGCU 501 TTCTGCTATTCCTTGTT 699 UAUUAUC TTAATTATGCTTATTAT CTTTTGGTTCT 8b_27970 CUCAACAUCAACCA 502 GTTTACAGTCATGTAC 700 UAUGUA TCAACATCAACCATAT GTAGTTGATGACC 8b_28043 AGCUAGAAAAUCA 503 GTATATTAGAGTAGGA 701 GCACCUU GCTAGAAAATCAGCAC CTTTAATTGAATT 8b_28219 UAGAGUAUCAUGA 504 TCTATGAAGACTTTTTA 702 CGUUCGU GAGTATCATGACGTTC GTGTTGTTTTAG 8b_27764 ACUUUCAUUAAUU 505 AGACAGAATGATTGAA 703 GACUUCU CTTTCATTAATTGACTT CTATTTGTGCTT 8b_27963 UCAUGUACUCAACA 506 GAATGTAGTTTACAGT 704 UCAACC CATGTACTCAACATCA ACCATATGTAGTT 8b_28104 AUUCAGUACAUCG 507 GGTTCTAAATCACCCA 705 AUAUCGG TTCAGTACATCGATAT CGGTAATTATACA 8b_28018 AUUCUAAAUGGUA 503 GTCCTATTCACTTCTAT 706 UAUUAGA TCTAAATGGTATATTA GAGTAGGAGCTA 8b_27892 ACAUGAAAUUUCU 509 GTCACGCCTAAACGAA 707 UGUUUUC CATGAAATTTCTTGTTT TCTTAGGAATCA E_26390 UGAGUCUUGUAAA 510 GCAATATTGTTAACGT 708 ACCUUCU GAGTCTTGTAAAACCT TCTTTTTACGTTT E_26391 GAGUCUUGUAAAA 511 CAATATTGTTAACGTG 709 CCUUCUU AGTCTTGTAAAACCTT CTTTTTACGTTTA E_26392 AGUCUUGUAAAAC 512 AATATTGTTAACGTGA 710 CUUCUUU GTCTTGTAAAACCTTCT TTTTACGTTTAC E_26468 UCUAAACGAACUA 513 TTCCTGATCTTCTGGTC 711 AAUAUUA TAAACGAACTAAATAT TATATTAGTTTT E_26321 UCUUGCUAGUUAC 514 TTGCTTTCGTGGTATTC 712 ACUAGCC TTGCTAGTTACACTAG CCATCCTTACTG E_26259 UUCGGAAGAGACA 515 TATGTACTCATTCGTTT 713 GGUACGU CGGAAGAGACAGGTAC GTTAATAGTTAA E_26460 UCUUCUGGUCUAA 516 TTCTAGAGTTCCTGATC 714 ACGAACU TTCTGGTCTAAACGAA CTAAATATTATA E_26284 GUUAAUAGCGUAC 517 ACAGGTACGTTAATAG 715 UUCUUUU TTAATAGCGTACTTCTT TTTCTTGCTTTC E_26314 GUGGUAUUCUUGC 518 CTTTTTCTTGCTTTCGT 716 UAGUUAC GGTATTCTTGCTAGTTA CACTAGCCATC E_26274 UACGUUAAUAGUU 519 TTCGGAAGAGACAGGT 717 AAUAGCG ACGTTAATAGTTAATA GCGTACTTCTTTT E_26260 UCGGAAGAGACAG 520 ATGTACTCATTCGTTTC 718 GUACGUU GGAAGAGACAGGTAC GTTAATAGTTAAT E_26308 GCUUUCGUGGUAU 521 GTACTTCTTTTTCTTGC 719 UCUUGCU TTTCGTGGTATTCTTGC TAGTTACACTA E_26411 UUUACGUUUACUC 522 TTGTAAAACCTTCTTTT 720 UCGUGUU TACGTTTACTCTCGTGT TAAAAATCTGA E_26265 AGAGACAGGUACG 523 CTCATTCGTTTCGGAA 721 UUAAUAG GAGACAGGTACGTTAA TAGTTAATAGCGT E_26279 UAAUAGUUAAUAG 524 AAGAGACAGGTACGTT 722 CGUACUU AATAGTTAATAGCGTA CTTCTTTTTCTTG E_26312 UCGUGGUAUUCUU 525 TTCTTTTTCTTGCTTTC 723 GCUAGUU GTGGTATTCTTGCTAGT TACACTAGCCA E_26464 CUGGUCUAAACGA 526 AGAGTTCCTGATCTTCT 724 ACUAAAU GGTCTAAACGAACTAA ATATTATATTAG E_26231 AUGAGUACGAACU 527 TGTAAGCACAAGCTGA 725 UAUGUAC TGAGTACGAACTTATG TACTCATTCGTTT E_26469 CUAAACGAACUAA 528 TCCTGATCTTCTGGTCT 726 AUAUUAU AAACGAACTAAATATT ATATTAGTTTTT E_26413 UACGUUUACUCUCG 529 GTAAAACCTTCTTTTTA 727 UGUUAA CGTTTACTCTCGTGTTA AAAATCTGAAT E_26281 AUAGUUAAUAGCG 530 GAGACAGGTACGTTAA 728 UACUUCU TAGTTAATAGCGTACT TCTTTTTCTTGCT E_26462 UUCUGGUCUAAAC 531 CTAGAGTTCCTGATCTT 729 GAACUAA CTGGTCTAAACGAACT AAATATTATATT M_26517 UUAGCCAUGGCAG 532 TTTGGAACTTTAATTTT 730 AUUCCAA AGCCATGGCAGATTCC AACGGTACTATT M_26522 CAUGGCAGAUUCCA 533 AACTTTAATTTTAGCC 731 ACGGUA ATGGCAGATTCCAACG GTACTATTACCGT M_26656 UUUUGUAUAUAAU 534 CCAACAGGAATAGGTT 732 UAAGUUA TTTGTATATAATTAAGT TAATTTTCCTCT M_26603 AUUCCUUACAUGG 535 AGTAATAGGTTTCCTA 733 AUUUGUC TTCCTTACATGGATTTG TCTTCTACAATT M_26572 UUGAACAAUGGAA 536 AGCTTAAAAAGCTCCT 734 CCUAGUA TGAACAATGGAACCTA GTAATAGGTTTCC M_27101 UGCUGCAUACAGUC 537 AGGTGACTCAGGTTTT 735 GCUACA GCTGCATACAGTCGCT ACAGGATTGGCAA M_27049 UUUCUUAUUACAA 538 CTACATCACGAACGCT 736 AUUGGGA TTCTTATTACAAATTGG GAGCTTCGCAGC M_26580 UGGAACCUAGUAA 539 AAGCTCCTTGAACAAT 737 UAGGUUU GGAACCTAGTAATAGG TTTCCTATTCCTT M_26733 UACAGAAUAAAUU 540 GTGCTTGCTGCTGTTTA 738 GGAUCAC CAGAATAAATTGGATC ACCGGTGGAATT M_26495 UUUUCUGUUUGGA 541 AAATATTATATTAGTTT 739 ACUUUAA TTCTGTTTGGAACTTTA ATTTTAGCCAT M_26608 UUACAUGGAUUUG 542 TAGGTTTCCTATTCCTT 740 UCUUCUA ACATGGATTTGTCTTCT ACAATTTGCCT M_26590 UAAUAGGUUUCCU 543 AACAATGGAACCTAGT 741 AUUCCUU AATAGGTTTCCTATTCC TTACATGGATTT M_26501 GUUUGGAACUUUA 544 TATATTAGTTTTTCTGT 742 AUUUUAG TTGGAACTTTAATTTTA GCCATGGCAGA M_26796 UGGCUCAGCUACUU 545 CTTGTAGGCTTGATGT 743 CAUUGC GGCTCAGCTACTTCAT TGCTTCTTTCAGA M_26570 CCUUGAACAAUGG 546 AGAGCTTAAAAAGCTC 744 AACCUAG CTTGAACAATGGAACC TAGTAATAGGTTT M_27124 UUGGCAACUAUAA 547 ACAGTCGCTACAGGAT 745 AUUAAAC TGGCAACTATAAATTA AACACAGACCATT M_26814 GCUUCUUUCAGACU 548 CTCAGCTACTTCATTGC 746 GUUUGC TTCTTTCAGACTGTTTG CGCGTACGCGT M_26694 UGGCCAGUAACUU 549 TTCCTCTGGCTGTTATG 747 UAGCUUG GCCAGTAACTTTAGCT TGTTTTGTGCTT M_27047 GCUUUCUUAUUAC 550 TGCTACATCACGAACG 748 AAAUUGG CTTTCTTATTACAAATT GGGAGCTTCGCA M_27153 UCCAGUAGCAGUG 551 TTAAACACAGACCATT 749 ACAAUAU CCAGTAGCAGTGACAA TATTGCTTTGCTT M_27163 GUGACAAUAUUGC 552 ACCATTCCAGTAGCAG 750 UUUGCUU TGACAATATTGCTTTG CTTGTACAGTAAG M_26473 ACGAACUAAAUAU 553 GATCTTCTGGTCTAAA 751 UAUAUUA CGAACTAAATATTATA TTAGTTTTTCTGT N_29327 UUGCUGAAUAAGC 554 AAAGATCAAGTCATTT 752 AUAUUGA TGCTGAATAAGCATAT TGACGCATACAAA N_29216 UUCGGAAUGUCGC 555 AGCGCTTCAGCGTTCT 753 GCAUUGG TCGGAATGTCGCGCAT TGGCATGGAAGTC N_28486 CGUUCCAAUUAACA 556 CCCTCGAGGACAAGGC 754 CCAAUA GTTCCAATTAACACCA ATAGCAGTCCAGA N_28755 UUCCUCAAGGAACA 557 CAATCGTGCTACAACT 755 ACAUUG TCCTCAAGGAACAACA TTGCCAAAAGGCT N_28585 CAGUCCAAGAUGG 558 TAAAATGAAAGATCTC 756 UAUUUCU AGTCCAAGATGGTATT TCTACTACCTAGG N_28565 GACGGUAAAAUGA 559 CGAATTCGTGGTGGTG 757 AAGAUCU ACGGTAAAATGAAAGA TCTCAGTCCAAGA N_28961 CUUGAGAGCAAAA 560 GACAGATTGAACCAGC 758 UGUCUGG TTGAGAGCAAAATGTC TGGTAAAGGCCAA N_29310 UCAAAGAUCAAGU 561 ACAAAGATCCAAATTT 759 CAUUUUG CAAAGATCAAGTCATT TTGCTGAATAAGC N_29177 CCGCAAAUUGCACA 562 GATTACAAACATTGGC 760 AUUUGC CGCAAATTGCACAATT TGCCCCCAGCGCT N_28261 CGAACAAACUAAA 563 TAGATTTCATCTAAAC 761 AUGUCUG GAACAAACTAAAATGT CTGATAATGGACC N_28476 GAGGACAAGGCGU 564 ACCTTAAATTCCCTCG 762 UCCAAUU AGGACAAGGCGTTCCA ATTAACACCAATA N_29130 UUGGGGACCAGGA 565 AAACCCAAGGAAATTT 763 ACUAAUC TGGGGACCAGGAACTA ATCAGACAAGGAA N_29455 UCUUCCUGCUGCAG 566 GCAAACTGTGACTCTT 764 AUUUGG CTTCCTGCTGCAGATTT GGATGATTTCTC N_29465 GCAGAUUUGGAUG 567 ACTCTTCTTCCTGCTGC 765 AUUUCUC AGATTTGGATGATTTC TCCAAACAATTG N_29132 GGGGACCAGGAAC 568 ACCCAAGGAAATTTTG 766 UAAUCAG GGGACCAGGAACTAAT CAGACAAGGAACT N_29148 UCAGACAAGGAAC 569 GGGACCAGGAACTAAT 767 UGAUUAC CAGACAAGGAACTGAT TACAAACATTGGC N_29291 GAUGACAAAGAUC 570 GGTGCCATCAAATTGG 768 CAAAUUU ATGACAAAGATCCAAA TTTCAAAGATCAA N_29342 AUUGACGCAUACA 571 TTGCTGAATAAGCATA 769 AAACAUU TTGACGCATACAAAAC ATTCCCACCAACA N_28589 CCAAGAUGGUAUU 572 ATGAAAGATCTCAGTC 770 UCUACUA CAAGATGGTATTTCTA CTACCTAGGAACT N_29133 GGGACCAGGAACU 573 CCCAAGGAAATTTTGG 771 AAUCAGA GGACCAGGAACTAATC AGACAAGGAACTG N_29057 CGUACUGCCACUAA 574 AAGCCTCGGCAAAAAC 772 AGCAUA GTACTGCCACTAAAGC ATACAATGTAACA N_28682 GGAGCCUUGAAUA 575 TGGGTTGCAACTGAGG 773 CACCAAA GAGCCTTGAATACACC AAAAGATCACATT orf1a_7249 UUUGGCAUAUAUU 576 AGTTGCAGAGTGGTTT 774 CUUUUCA TTGGCATATATTCTTTT CACTAGGTTTTT orf1a_1066 UGGGGAAUGUCCA 577 ATTTGACACCTTCAAT 775 AAUUUUG GGGGAATGTCCAAATT TTGTATTTCCCTT orf1a_11197 UGCCACUGUAGCUU 578 TTTGTTACCTTCTCTTG 776 AUUUUA CCACTGTAGCTTATTTT AATATGGTCTA orf1a_13292 UGUGACUUAAAAG 579 AATCCTAAAGGATTTT 777 GUAAGUA GTGACTTAAAAGGTAA GTATGTACAAATA orf1a_12931 UCCUAAAGUGAAG 580 AGACACACCTAAAGGT 778 UAUUUAU CCTAAAGTGAAGTATT TATACTTTATTAA orf1a_4598 CUUGUUACAAUGCC 581 GATCTAAATGAAACTC 779 ACUUGG TTGTTACAATGCCACTT GGCTATGTAACA orf1a_12515 UGUGAUGGUACAA 582 ACATATAAAAATACGT 780 CAUUUAC GTGATGGTACAACATT TACTTATGCATCA orf1a_9060 AUUGUUAUGAUAC 583 GTAAGCCAGTACCATA 781 CAAUGUA TTGTTATGATACCAAT GTACTAGAAGGTT orf1a_1286  UGCGAAUUUUGUG 584 TTTGTTAAAGCCACTT 782 GCACUGA GCGAATTTTGTGGCAC TGAGAATTTGACT orf1a_9707 AUUUCCACAAAGC 585 GCTTATATCATTTGTAT 783 AUUUCUA TTCCACAAAGCATTTC TATTGGTTCTTT orf1a_5110 UAAAACAUUUUAU 586 TAATTCACATGAAGGT 784 GUUUUAC AAAACATTTTATGTTTT ACCTAATGATGA orf1a_9041 UCUGGUAAGCCAG 587 ATTTTTAAAGATGCTTC 785 UACCAUA TGGTAAGCCAGTACCA TATTGTTATGAT orf1a_11687 CUUGGUGUUUAUG 588 TACTTTAGACTGACTCT 786 AUUACUU TGGTGTTTATGATTACT TAGTTTCTACA orf1a_12215 UUGAAUGUGGCUA 589 AAGTTGAAGAAGTCTT 787 AAUCUGA TGAATGTGGCTAAATC TGAATTTGACCGT orf1a_11254 UAUGACAUGGUUG 590 TTGGGTGATGCGTATT 788 GAUAUGG ATGACATGGTTGGATA TGGTTGATACTAG orf1a_6963 UUUUACUAUUAAG 591 ATATTATAATTTGGTTT 789 UGUUUGC TTACTATTAAGTGTTTG CCTAGGTTCTT orf1a_2969 AUGGCUACAUACU 592 TTAGATGAGTGGAGTA 790 ACUUAUU TGGCTACATACTACTT ATTTGATGAGTCT orf1a_5449 UAGAGAAACAAUG 593 TGAGTTAGGTGATGTT 791 AGUUACU AGAGAAACAATGAGTT ACTTGTTTCAACA orf1a_5238 ACCCACAAGUUAAU 594 CTAAAAAGTGGAAATA 792 GGUUUA CCCACAAGTTAATGGT TTAACTTCTATTA orf1a_8059 UUCAUCAACUUUU 595 TTACGTTAATACGTTTT 793 AACGUAC CATCAACTTTTAACGT ACCAATGGAAAA orf1a_12276 UGGCUGAUCAAGC 596 GTAAGTTGGAAAAGAT 794 UAUGACC GGCTGATCAAGCTATG ACCCAAATGTATA orf1a_4097 UUCUUAAAGAAAG 597 GACATTGACATCACTT 795 AUGCUCC TCTTAAAGAAAGATGC TCCATATATAGTG orf1ab_19120 UAUAAAAUAGAAG 598 TGTAGTGACAAAGCTT 796 AAUUAUU ATAAAATAGAAGAATT ATTCTATTCTTAT orf1ab_17193 GAAGGCAUUAAAA 599 TGATGCACTATGTGAG 797 UAUUUGC AAGGCATTAAAATATT TGCCTATAGATAA orf1ab_17034 UGCAAAUUAUCAA 600 GTTTTCTAGCAATGTTG 798 AAGGUUG CAAATTATCAAAAGGT TGGTATGCAAAA orf1ab_19748 UUGAAAAUAAAAC 601 TTGATGTAGAATTGTTT 799 AACAUUA GAAAATAAAACAACAT TACCTGTTAATG orf1ab_17980 UAUGACAAGUUGC 602 TCTGATAGAGACCTTT 800 AAUUUAC ATGACAAGTTGCAATT TACAAGTCTTGAA orf1ab_21482 UUAGUAAAGGUAG 603 TGATTTTATCTCTTCTT 801 ACUUAUA AGTAAAGGTAGACTTA TAATTAGAGAAA orf1ab_13842 UGAUGAAGGUAAU 604 TGCTTTAAGGCATTTTG 802 UGUGACA ATGAAGGTAATTGTGA CACATTAAAAGA orf1ab_19071 UGAUGUAGAAUGG 605 GTGTGTACCTCAAGCT 803 AAGUUCU GATGTAGAATGGAAGT TCTATGATGCACA orf1ab_13878 UGUCACAUACAAU 606 ATTAAAAGAAATACTT 804 UGUUGUG GTCACATACAATTGTT GTGATGATGATTA orf1ab_16555 AAUGCAAUUGCAA 607 AATGTTACTGACTTTA 805 CAUGUGA ATGCAATTGCAACATG TGACTGGACAAAT orf1ab_17284 UCAACAUUAGAAC 608 AAATTCAAAGTGAATT 806 AGUAUGU CAACATTAGAACAGTA TGTCTTTTGTACT orf1ab_17852 CAGAAUAUGACUA 609 ATTCATCACAGGGCTC 807 UGUCAUA AGAATATGACTATGTC ATATTCACTCAAA orf1ab_17671 GUUAUCACGCAUG 610 ATGTTTTATAAGGGTG 808 AUGUUUC TTATCACGCATGATGT TTCATCTGCAATT orf1ab_21331 CAUGCAAAUUACA 611 GATGGTTATGTCATGC 809 UAUUUUG ATGCAAATTACATATT TTGGAGGAATACA orf1ab_18612 GUUGACAUCUAUG 612 GGCACATGGCTTTGAG 810 AAGUAUU TTGACATCTATGAAGT ATTTTGTGAAAAT orf1ab_19345 UUUGUUAAUUUAA 613 TTTGATAAAAGTGCTT 811 AACAAUU TTGTTAATTTAAAACA ATTACCATTTTTC orf1ab_15409 AAUGAGUGUGCUC 614 TTCTATAGATTAGCTA 812 AAGUAUU ATGAGTGTGCTCAAGT ATTGAGTGAAATG orf1ab_19617 UCAGAGUUUAGAA 615 CACTTTTACAAGACTT 813 AAUGUGG CAGAGTTTAGAAAATG TGGCTTTTAATGT orf1ab_21444 AAAAGAAGGUCAA 616 TGCTGTTATGTCTTTA 814 AUCAAUG AAAGAAGGTCAAATCA ATGATATGATTTT orf1ab_20511 AUUACUUGAUGAU 617 TTCTGTTATTGATTTA 815 UUUGUUG TTACTTGATGATTTTG TTGAAATAATAAA orf1ab_21174 AGAACAUUCUUGG 618 GGCTATAAAGATAACA 816 AAUGCUG GAACATTCTTGGAATG CTGATCTTTATAA orf1ab_19732 GUUGAUGUAGAAU 619 ACAAAAGTTGATGGTG 817 UGUUUGA TTGATGTAGAATTGTT TGAAAATAAAACA S_24473 GGUGCAAUUUCAA 620 CTTAGCTCCAATTTTGG 818 GUGUUUU TGCAATTTCAAGTGTTT TAAATGATATC S_22368 AUCUUCAACCUAGG 621 CTTATTATGTGGGTTAT 819 ACUUUU CTTCAACCTAGGACTT TTCTATTAAAAT S_24635 GCUGCUACUAAAA 622 GCTTCTGCTAATCTTGC 820 UGUCAGA TGCTACTAAAATGTCA GAGTGTGTACTT S_21952 CUGUGAAUUUCAA 623 TGTTGTTATTAAAGTCT 821 UUUUGUA GTGAATTTCAATTTTGT AATGATCCATT S_23698 UGCCAUACCCACAA 624 CTCTAATAACTCTATTG 822 AUUUUA CCATACCCACAAATTT TACTATTAGTGT S_23936 CCACCAAUUAAAGA 625 CAAATTTACAAAACAC 823 UUUUGG CACCAATTAAAGATTT TGGTGGTTTTAAT S_22807 UGGAAAGAUUGCU 626 CGCTCCAGGGCAAACT 824 GAUUAUA GGAAAGATTGCTGATT ATAATTATAAATT S_21938 GUUGUUAUUAAAG 627 AATAACGCTACTAATG 825 UCUGUGA TTGTTATTAAAGTCTGT GAATTTCAATTT S_22138 UGUGUUUAAGAAU 628 AAATCTTAGGGAATTT 826 AUUGAUG GTGTTTAAGAATATTG ATGGTTATTTTAA S_24828 UUCCUCGUGAAGG 629 ATGGAAAAGCACACTT 827 UGUCUUU TCCTCGTGAAGGTGTC TTTGTTTCAAATG S_23315 CUUGACAUUACACC 630 CAGACACTTGAGATTC 828 AUGUUC TTGACATTACACCATG TTCTTTTGGTGGT S_25376 UACACAUAAACGA 631 GGAGTCAAATTACATT 829 ACUUAUG ACACATAAACGAACTT ATGGATTTGTTTA S_22259 AUUAACAUCACUA 632 GATTTGCCAATAGGTA 830 GGUUUCA TTAACATCACTAGGTT TCAAACTTTACTT S_22129 UAGGGAAUUUGUG 633 TAATTTCAAAAATCTT 831 UUUAAGA AGGGAATTTGTGTTTA AGAATATTGATGG S_24254 UUUGCUAUGCAAA 634 GCATTACAAATACCAT 832 UGGCUUA TTGCTATGCAAATGGC TTATAGGTTTAAT S_22276 UCAAACUUUACUU 635 TAACATCACTAGGTTT 833 GCUUUAC CAAACTTTACTTGCTTT ACATAGAAGTTA S_23270 GCUGACACUACUGA 636 TTTGGCAGAGACATTG 834 UGCUGU CTGACACTACTGATGC TGTCCGTGATCCA S_25235 AUUGCCAUAGUAA 637 TTTATAGCTGGCTTGAT 835 UGGUGAC TGCCATAGTAATGGTG ACAATTATGCTT S_23842 UACACAAUUAAACC 638 ATATGGCAGTTTTTGT 836 GUGCUU ACACAATTAAACCGTG CTTTAACTGGAAT S_23307 UUGAGAUUCUUGA 639 GTGATCCACAGACACT 837 CAUUACA TGAGATTCTTGACATT ACACCATGTTCTT S_21808 UGUCCUACCAUUUA 640 GAGGTTTGATAACCCT 838 AUGAUG GTCCTACCATTTAATG ATGGTGTTTATTT S_23935 ACCACCAAUUAAAG 641 ACAAATTTACAAAACA 839 AUUUUG CCACCAATTAAAGATT TTGGTGGTTTTAA

TABLE 8 Top SARS-CoV-2 based on homology.   SEQ SEQ ID ID Sequence ID 20 nt Sequence NO: 45 nt Gene Region NO: E_26313 CGUGGUAUUCU  64 TCTTTTTCTTGCTTTCGTGGTATT 172 UGCUAGUUA CTTGCTAGTTACACTAGCCAT 8b_27986 UGUAGUUGAUG  97 TCAACATCAACCATATGTAGTTG 205 ACCCGUGUC ATGACCCGTGTCCTATTCACTT 7a27720 UAUAACACUUU  93 TGCGGCAATAGTGTTTATAACAC 201 GCUUCACAC TTTGCTTCACACTCAAAAGAAA orf1ab_14080 GGUAACUGGUA  11 AATCAAGATCTCAATGGTAACTG 119 UGAUUUCGG GTATGATTTCGGTGATTTCATA M_26581 GGAACCUAGUA  72 AGCTCCTTGAACAATGGAACCTA 180 AUAGGUUUC GTAATAGGTTTCCTATTCCTTA 522820 GAUUAUAAUUA  38 ACTGGAAAGATTGCTGATTATAA 146 UAAAUUACC TTATAAATTACCAGATGATTTT orf1ab_21391 UUUGACAUGAG  22 TCTTCCTATTCTTTATTTGACATG 130 UAAAUUUCC AGTAAATTTCCCCTTAAATTA orf1ab_17107 UUUGCUAUUGG  16 ACTGGTAAGAGTCATTTTGCTAT 124 CCUAGCUCU TGGCCTAGCTCTCTACTACCCT orf1ab_20892 UGCACCAGGUA  21 TTCTGATAAAGGAGTTGCACCAG 129 CAGCUGUUU GTACAGCTGTTTTAAGACAGTG orf1ab_20497 UCUGUUAUUGA  20 TCTAAGTGTGTGTGTTCTGTTATT 128 UUUAUUACU GATTTATTACTTGATGATTTT N_28655 GACGGCAUCAU 108 TATGGTGCTAACAAAGACGGCAT 216 AUGGGUUGC CATATGGGTTGCAACTGAGGGA 524289 UGGAGUUACAC  45 TAGGTTTAATGGTATTGGAGTTA 153 AGAAUGUUC CACAGAATGTTCTCTATGAGAA M_26624 UCUACAAUUUG  74 TACATGGATTTGTCTTCTACAATT 182 CCUAUGCCA TGCCTATGCCAACAGGAATAG orf1a_7643 GCUGGUAGUAC  28 TGTGATACATTCTGTGCTGGTAG 136 AUUUAUUAG TACATTTATTAGTGATGAAGTT orf1a_12932  CCUAAAGUGAA  34 GACACACCTAAAGGTCCTAAAGT 142 GUAUUUAUA GAAGTATTTATACTTTATTAAA E_26305 CUUGCUUUCGU  63 AGCGTACTTCTTTTTCTTGCTTTC 171 GGUAUUCUU GTGGTATTCTTGCTAGTTACA M_26602 UAUUCCUUACA  73 TAGTAATAGGTTTCCTATTCCTTA 181 UGGAUUUGU CATGGATTTGTCTTCTACAAT 3a_25630 GUUUGCAACUU  48 AAGGGTGTTCACTTTGTTTGCAA 156 GCUGUUGUU CTTGCTGTTGTTGTTTGTAACA E_26455 CCUGAUCUUCU  67 AATTCTTCTAGAGTTCCTGATCTT 175 GGUCUAAAC CTGGTCTAAACGAACTAAATA E_26463 UCUGGUCUAAA  68 TAGAGTTCCTGATCTTCTGGTCTA 176 CGAACUAAA AACGAACTAAATATTATATTA 522550 CCUAAUAUUAC  37 TCTATTGTTAGATTTCCTAATATT 145 AAACUUGUG ACAAACTTGTGCCCTTTTGGT orf1a_8744 UUUGCUAACAA  31 TCTACAGATACTTGTTTTGCTAAC 139 ACAUGCUGA AAACATGCTGATTTTGACACA orf1ab_17370 GGCCACAAAUU  17 TGATGAAATTTCAATGGCCACAA 125 AUGAUUUGA ATTATGATTTGAGTGTTGTCAA 7a_27633 AUCAGUUUCAC  88 TCAGTTACGTGCCAGATCAGTTT 196 CUAAACUGU CACCTAAACTGTTCATCAGACA M_27014 GCCUAAAGAAA  79 TGACATCAAGGACCTGCCTAAAG 187 UCACUGUUG AAATCACTGTTGCTACATCACG S_25375 UUACACAUAAA  46 AGGAGTCAAATTACATTACACAT 154 CGAACUUAU AAACGAACTTATGGATTTGTTT 7a_27715 GUGUUUAUAAC  92 ATTGTTGCGGCAATAGTGTTTAT 200 ACUUUGCUU AACACTTTGCTTCACACTCAAA

TABLE 9 Antisense oligonucleotides targeting viral factors SEQ ID Oligo ID Sequence NO: orf1a_416 ACTTCTACTAAGCCAC 840 orf1a_2290 AAGCTTAAAGAATGTC 841 orf1a_8744-1 CAGCATGTTTGTTAGC 842 orf1a_8744-2 GCATGTTTGTTAGCAA 843 orf1a_9679-1 AAGCAATTGTTATCCA 844 orf1a_9679-2 TAAGCAATTGTTATCC 845 orf1ab_14361 GTTTGCACAATGCAGA 846 orf1ab_17107 AGAGCTAGGCCAATAG 847 orf1ab_18025 AGCTTGTAAAGTTGCC 848 orf1ab_20892-1 CAGCTGTACCTGGTGC 849 orf1ab_20892-2 ACAGCTGTACCTGGTG 850 orf1ab_20892-3 AACAGCTGTACCTGGT 851 S_22223-1 AATGGTTCTAAAGCCG 852 S_22223-2 CAATGGTTCTAAAGCC 853 S_23174 TGAAGTTGAAATTGAC 854 S_23774 CCACAAATGTACATTG 855 S_23778 CAATGTACATTTGTGG 856 S_25375-1 AGTTCGTTTATGTGTA 857 S_25375-2 TAAGTTCGTTTATGTG 858 3a_25717 GAAGTAGACTAAAGCA 859 3a_25914 AATAGGACTTGTTGTG 860 3a_25992 AACTGTGTAATACAAC 861 3a_26018-1 ACAGCTGGTAATAGTC 862 3a_26018-2 CAGCTGGTAATAGTCT 863 3a_26018-3 TACAGCTGGTAATAGT 864 E_26258 TACCTGTCTCTTCCGA 865 E_26261 TAACGTACCTGTCTCT 866 E_26369-1 AACAATATTGCAGCAG 867 E_26369-2 TAACAATATTGCAGCA 868 E_26374-1 CGTTAACAATATTGCA 869 E_26374-2 CTCACGTTAACAATAT 870 M_26581-1 ACCTATTACTAGGTTC 871 M_26581-2 AAACCTATTACTAGGT 872 M_26602-1 ACAAATCCATGTAAGG 873 M_26602-2 CAAATCCATGTAAGGA 874 M_26624 TGGCATAGGCAAATTG 875 M_26717 GTAAACAGCAGCAAGC 876 7a_27455 GTACCTCTAACACACT 877 7a_27553 AGCAAGTCAGTGCAAA 878 7a_27565-1 ATTGAGTGCTAAAGCA 879 7a_27565-2 GCAAATTGAGTGCTAA 880 7a_27705 AAACACTATTGCCGCA 881 7a_27720 GTGTGAAGCAAAGTGT 882 8b_27940 AAACTACATTCTTGGT 883 8b_28002 AGAAGTGAATAGGACA 884 8b_28091-1 TGAATGGGTGATTTAG 885 8b_28091-2 GAATGGGTGATTTAGA 886 8b_28119 AAACTGTATAATTACC 887 8b_28163 CCCAATTTAGGTTCCT 888 N_28655 ACCCATATGATGCCGT 889 N_28945-1 CAAGCTGGTTCAATCT 890 N_28945-2 GCTGGTTCAATCTGTC 891 N_29141-1 CCTTGTCTGATTAGTT 892 N_29141-2 CTTGTCTGATTAGTTC 893 N_29307 AATGACTTGATCTTTG 894

An alignment of siRNA and ASO selected for synthesis to six closely-related CoVs using the novel algorithm is shown in FIG. 3 . Aligned genome regions of CoVs are shaded based on homology with darker coloring indicating higher homology with respect to SARS-CoV-2. The siRNA position is indicated on the top. Per position percent homology of SARS-CoV-2 to the six related CoVs is plotted on the bottom. SiRNA with low homology scores of 59 are shown in FIG. 3A. SiRNA with a high homology scores of score of 78 are shown in FIG. 3B. Gaps in alignment are indicated with dashes (-).

SiRNA and ASO target selections were based on the ability to target many SARS-CoV-2 genomes from patient isolates. siRNAs and ASOs were selected to target regions of the 9 selection genes with low mutation rates in other coronaviruses (FIG. 4A). The proportion of SARS-CoV-2 variants from patient isolates targeted by all selected siRNAs is plotted at the bottom. siRNAs and ASOs were selected to target regions of the 9 selection genes with low mutation rates in other coronaviruses (see previous figure), which resulted in selecting siRNAs that together target all isolates from SARS-CoV-infected patients, with >90% of siRNAs and ASOs selected targeting >95% of these genomes. The proportion of SARS-CoV-2 variants from patient isolates targeted by all selected siRNAs is plotted at the bottom. The coloured gradient indicates proportion of variant genomes targeted, with red indicating siRNAs that target fewer variants (with the lowest value being 77% of all variants targeted) and white indicating higher number of genomes targeted. Arrows in the gene diagram indicate siRNA and ASO target positions in the SARS-CoV2 genome and are coloured based on proportion of SARS-CoV-2 variants targeted and correlate with the colored gradient in the bottom plot. Dark red arrows indicate siRNAs that target <85% of genomes from patient isolates, while white and light pink arrows indicate siRNAs that target >90% of genomes SARS-CoV-2 isolates patients.

The scoring scheme methodology resulted in the selection of siRNAs that target all 708 SARS-CoV2 patient isolates available at the time of design with >95% of siRNAs (103 siRNAs) selected targeting more than 90% of the SARS-CoV2 genomes obtained from patient isolates. Furthermore, >60% of siRNAs (66 siRNAs) selected target more than 97% of these patient isolates.

SiRNAs targeting different genes in the SARS-CoV2 genome were tested for silencing efficacy of the orfla, 3a, 7a, orflab, E, gene 8b, S, M and N genes (FIG. 5 , FIG. 7 ). For each target, at least 3 siRNAs were identified that that reduced target mRNA expression below 75% compared to untreated controls. siRNAs were tested in Hela cells and silencing was assessed using the psi-check reporter system, using an siRNA concentration of 1.5 uM and an assessment timepoint of 72 hours. Likewise, ASOs targeting the different genes in the SARS-CoV2 genome were tested for silencing efficacy, as depicted in FIG. 6 and FIG. 8 . For each target, at least 3 ASOs were identified that reduced target mRNA expression below 75% compared to untreated controls. SiRNAs targeting the orfla, 3a, 7a, orflab, E, gene 8b, S, M and N genes in the SARS-CoV2 genome were subsequently tested for silencing efficacy in 8-point dose response studies. Each siRNA showed potent and efficacious target silencing with IC₅₀ values in the low nanomolar range.

FIG. 10 depicts a schematic showing genes comprising genome and their functions. Comprised of structural and non-structural genes. Non-structural genes undergo primary translation while structural and accessory proteins are translated from sub-genomic mRNAs. The secondary structure of sub-genomic mRNAs may enhance targetability by siRNAs.

Development and optimization of siRNA cocktails targeting SARS-CoV2. Like other RNA viruses, SARS-CoV2 mutates. Therefore, multiple siRNAs in cocktails are necessary to minimize the chances of mutant development. Several siRNA cocktails were developed targeting g+ strand only, g+ strand and terminal 3′UTR (region shared by all secondary mRNA variants), as well as a variety of other combinations. The cocktails are screened in SARS-CoV-2 infected VERO6 cells to define optimal siRNA combinations against the live virus. The strategy of targeting the + strand (orfla and orflab with S and N protein) is particularly novel and active in blocking SARS-CoV-2 infection. The cocktails are combinations of at least 3 siRNAs targeting different regions of the viral genome. Table 10 shows the compositions of the various cocktails tested.

TABLE 10 List of siRNA combinations for targeting SARS-COV-2 Combo Code name Strategy Compounds (Sequence IDs) 1 Cosmopolitan Replication 416 9679 21391 2 Screwdriver Replication 416 8744 21391 3 Long Island Replication 9679 8744 21391 4 Negroni Rep/Immun 416 9679 27565 5 Old Fashioned Rep/Immun 416 21391 27565 6 Manhattan Rep/Immun 21391 27656 27751 7 Moscow Mule Rep/capsid 416 23174 26305 8 Daiquiri Rep/capsid 9679 23174 29293 9 Martini Rep/capsid 21391 26305 29293 10 Jaegger Bomb Immun/capsid 23174 26470 27565 11 Bloody Mary Immun/capsid 23174 27123 27656 12 White Russian Immun/capsid 23174 27032 27751 13 Mojito Rep/imm/cap 416 26305 27565 14 Salty Dog Rep/imm/cap 9679 26369 27656 15 Hanky Panky Rep/imm/cap 21391 27032 27751

The validation and determination of IC₅₀ values for siRNA cocktails targeting SARS-CoV-2 genes are depicted in FIG. 11 . SiRNA cocktails targeting different genes in the SARS-CoV2 genome were tested for silencing efficacy in 8-point dose response studies. Each siRNA showed potent and efficacious target silencing with IC₅₀ values in the low nanomolar range. Results for replication cocktails are shown in FIG. 11A, for Replication/Immuno cocktails in FIG. 11B, for Replication/Capsid cocktails in FIG. 11C, for Immuno/Capsid cocktails in FIG. 11D, and for Replication/Immuno/Capsid cocktails in FIG. 11E.

Example 2. Targeting of Human Genes

SiRNAs targeting of ACE2, FURIN, TMPRSS2, IL-6, and IL-6 Receptor (IL-6R) can be used alone or in combination with siRNAs targeting SARS-CoV2 for comprehensive treatment of SARS-CoV-2 treatment. Hyper functional, fully chemical stabilized siRNAs were identified targeting these a selection of these host cell genes involved in infection and spread.

Studies of ACE2 knock out mice show some toxicity in the heart and muscle, thus limiting the use of traditional small molecules and antibodies that do not differentiate between tissues. EPA-conjugates have no functional delivery to muscle and heart, and thus might be a better option for ACE2 modulation. In addition, local intratracheal delivery of di-valent compounds results in minimal heart and muscle delivery, representing a very powerful option for modulation of host genes, where lung-selective targeting with minimized overall exposure is required.

Host target 45 nucleotide gene regions and 20 nucleotide target regions are summarized in Table 11A-11D and Table 12A-12E.

TABLE 11A Host target TMPRSS-20 nucleotide targets and 45 nucleotide gene target regions SEQUENCE SEQ ID SEQ ID ID 20 nt Sequence NO: 45 nt Gene Region NO: Species TMPRSS UCCCGCAU  895 AGTGACTCATGTTCAT 1095 mouse 2_924 GGUGGUUU CCCGCATGGTGGTTTC CUUU TTTGCGCTGTATA TMPRSS UACCACAG  896 CTCTATAAAAAACTCT 1096 mouse 2_903 UGACUCAU ACCACAGTGACTCATG GUUC TTCATCCCGCATG TMPRSS UGAGCUCA  897 TTATGAAGCTGAATGT 1097 mouse 2_868 GGCAACGU GAGCTCAGGCAACGT UGAC TGACCTCTATAAAA TMPRSS UCUUCAAA  898 CACAGTGATGCCTGTT 1098 human 2_866 AGCAGUGG CTTCAAAAGCAGTGGT UUUC TTCTTTACGCTGT TMPRSS UGUGAGCU  899 CTTTATGAAGCTGAAT 1099 mouse 2_866 CAGGCAAC GTGAGCTCAGGCAAC GUUG GTTGACCTCTATAA TMPRSS ACCACAGU  900 TCTATAAAAAACTGTA 1100 human 2_849 GAUGCCUG CCACAGTGATGCCTGT UUCU TCTTCAAAAGCAG TMPRSS CCGGCAAU  901 AACTGAACACAAGTG 1101 human 2_819 GUCGAUAU CCGGCAATGTCGATAT CUAU CTATAAAAAACTGT TMPRSS AGUGCCGG  902 ATGAAACTGAACACA 1102 human 2_815 CAAUGUCG AGTGCCGGCAATGTC AUAU GATATCTATAAAAAA TMPRSS AAAGACAU  903 GGGAGAGCAGCATGT 1103 mouse 2_783 GGGAUACA AAAGACATGGGATAC AGAA AAGAACAATTTTTAT TMPRSS UUUACUCU  904 GCTATAAGAATAATTT 1104 human 2_753 AGCCAAGG TTACTCTAGCCAAGGA AAUA ATAGTGGATGACA TMPRSS GCUUCAUC  905 GTCTCTACGGACAAA 1105 mouse 2_691 CUCCAGGU GCTTCATCCTCCAGGT UUAC TTACTCATCTCAGA TMPRSS ACGGACAA  906 GTTGTGTTCGTCTCTA 1106 mouse 2_682 AGCUUCAU CGGACAAAGCTTCATC CCUC CTCCAGGTTTACT TMPRSS UGGGUCUU  907 GTCTGAGATGGAGTGT 1107 mouse 2_587 CAGGCACA GGGTCTTCAGGCACAT UGCA GCATCAGCTCTTC TMPRSS UCUGGGAC  908 TCTTGCTTTGGAGGTT 1108 mouse 2_550 AGCAACUG CTGGGACAGCAACTG UUCU TTCTACGTCTGAGA TMPRSS ACCUGCAU  909 TGCGACTCCTCAGGTA 1109 human 2_545 CAACCCCU CCTGCATCAACCCCTC CUAA TAACTGGTGTGAT TMPRSS CUUGCUUU  910 TGCTGTGGCTGCTGTC 1110 mouse 2_536 GGAGGUUC TTGCTTTGGAGGTTCT UGGG GGGACAGCAACTG TMPRSS AUGGGCAG  911 CTACTCTGGAAGTTCA 1111 human 2_497 CAAGUGCU TGGGCAGCAAGTGCT CCAA CCAACTCTGGGATA TMPRSS UAAGAAAU  912 GTGCACCTCAAAGTCT 1112 mouse 2_470 CGCUGUGU AAGAAATCGCTGTGTT UUAG TAGCCCTCGCCCT TMPRSS CUCAAAGU  913 AGGAGCACTGTGCAC 1113 mouse 2_461 CUAAGAAA CTCAAAGTCTAAGAA UCGC ATCGCTGTGTTTAGC TMPRSS UAAGAAAG  914 GTGCACCTCAAAGACT 1114 human 2_415 CACUGUGC AAGAAAGCACTGTGC AUCA ATCACCTTGACCCT TMPRSS CUCAACAU  915 GATTACAACGCAAGC 1115 mouse 2_410 CUGUCAUC CTCAACATCTGTCATC CACA CACACACATCCCAA TMPRSS AUCAUGCA  916 ATTGTGAAAATGAAT 1116 human 2_3204 AAUAAAUU ATCATGCAAATAAATT AUGC ATGCAATTTTTTTT TMPRSS UAUCAUGC  917 AATTGTGAAAATGAA 1117 human 2_3203 AAAUAAAU TATCATGCAAATAAAT UAUG TATGCAATTTTTTT TMPRSS GUGAAAAU  918 CTGTAAAGTTCAATTG 1118 human 2_3192 GAAUAUCA TGAAAATGAATATCAT UGCA GCAAATAAATTAT TMPRSS UUGUGAAA  919 AACTGTAAAGTTCAAT 1119 human 2_3190 AUGAAUAU TGTGAAAATGAATATC CAUG ATGCAAATAAATT TMPRSS UUCAAUUG  920 TTTTAAACTGTAAAGT 1120 human 2_3185 UGAAAAUG TCAATTGTGAAAATGA AAUA ATATCATGCAAAT TMPRSS GUUCAAUU  921 TTTTTAAACTGTAAAG 1121 human 2_3184 GUGAAAAU TTCAATTGTGAAAATG GAAU AATATCATGCAAA TMPRSS UGUAAAGU  922 GTATCTTTTTTAAACT 1122 human 2_3178 UCAAUUGU GTAAAGTTCAATTGTG GAAA AAAATGAATATCA TMPRSS AACUGUAA  923 TTTGTATCTTTTTTAA 1123 human 2_3175 AGUUCAAU ACTGTAAAGTTCAATT UGUG GTGAAAATGAATA TMPRSS UUAAACUG  924 TTTTTTGTATCTTTTTT 1124 human 2_3172 UAAAGUUC AAACTGTAAAGTTCA AAUU ATTGTGAAAATGA TMPRSS UUUAAACU  925 TTTTTTTGTATCTTTTT 1125 human 2_3171 GUAAAGUU TAAACTGTAAAGTTCA CAAU ATTGTGAAAATG TMPRSS GUGAACAA  926 CCCCTTCTTATTTATG 1126 human 2_3139 CUGUUUGU TGAACAACTGTTTGTC CUUU TTTTTTTGTATCT TMPRSS AUGUGAAC  927 TGCCCCTTCTTATTTA 1127 human 2_3137 AACUGUUU TGTGAACAACTGTTTG GUCU TCTTTTTTTGTAT TMPRSS UAUGUGAA  928 TTGCCCCTTCTTATTT 1128 human 2_3136 CAACUGUU ATGTGAACAACTGTTT UGUC GTCTTTTTTTGTA TMPRSS UUAUGUGA  929 ATTGCCCCTTCTTATT 1129 human 2_3135 ACAACUGU TATGTGAACAACTGTT UUGU TGTCTTTTTTTGT TMPRSS AUUUAUGU  930 TTATTGCCCCTTCTTA 1130 human 2_3133 GAACAACU TTTATGTGAACAACTG GUUU TTTGTCTTTTTTT TMPRSS AAAUAUAU  931 TGAGATTCCACTGTGA 1131 mouse 2_3130 GAAUAAAG AATATATGAATAAAG UAUA TATATAATTCTTTT TMPRSS UCUUAUUU  932 TTCTTTATTGCCCCTTC 1132 human 2_3129 AUGUGAAC TTATTTATGTGAACAA AACU CTGTTTGTCTTT TMPRSS AUUGCCCC  933 ACGTCTTCCTTCTTTA 1133 human 2_3120 UUCUUAUU TTGCCCCTTCTTATTT UAUG ATGTGAACAACTG TMPRSS GAUUCCAC  934 TGTTCTGAGCTGTGAG 1134 mouse 2_3118 UGUGAAAU ATTCCACTGTGAAATA AUAU TATGAATAAAGTA TMPRSS UCUGAGCU  935 CTGCTTTGTGTCTGTT 1135 mouse 2_3106 GUGAGAUU CTGAGCTGTGAGATTC CCAC CACTGTGAAATAT TMPRSS UGUGUCUG  936 GGTTATTTCCTGCTTT 1136 mouse 2_3097 UUCUGAGC GTGTCTGTTCTGAGCT UGUG GTGAGATTCCACT TMPRSS UAAUGGUG  937 TCCTAAAAGGTGTTGT 1137 human 2_3094 AAAACGUC AATGGTGAAAACGTC UUCC TTCCTTCTTTATTG TMPRSS GUAAUGGU  938 ATCCTAAAAGGTGTTG 1138 human 2_3093 GAAAACGU TAATGGTGAAAACGT CUUC CTTCCTTCTTTATT TMPRSS CCUGCUUU  939 GTTGGTTGGTTATTTC 1139 mouse 2_3090 GUGUCUGU CTGCTTTGTGTCTGTT UCUG CTGAGCTGTGAGA TMPRSS UUGGUUAU  940 TGTCTTTGCTGTTGGT 1140 mouse 2_3080 UUCCUGCU TGGTTATTTCCTGCTT UUGU TGTGTCTGTTCTG TMPRSS ACAUCCUA  941 CTTGCTCCCCAAGACA 1141 human 2_3076 AAAGGUGU CATCCTAAAAGGTGTT UGUA GTAATGGTGAAAA TMPRSS CUGUGCAC  942 ATTTGCAGGATCTGTC 1142 human 2_2994 AUGCCUCU TGTGCACATGCCTCTG GUAG TAGAGAGCAGCAT TMPRSS GCUGUAAG  943 GGGACCTTCTTAGATG 1143 mouse 2_2988 GUACCUAC CTGTAAGGTACCTACA AUAC TACAGACTAAATG TMPRSS UUUGCAGG  944 AGTCATGCAATCCCAT 1144 human 2_2980 AUCUGUCU TTGCAGGATCTGTCTG GUGC TGCACATGCCTCT TMPRSS AAUCCCAU  945 AATGGAAAGTCATGC 1145 human 2_2973 UUGCAGGA AATCCCATTTGCAGGA UCUG TCTGTCTGTGCACA TMPRSS GGGACCUU  946 CCTCATGCGTCCTCTG 1146 mouse 2_2973 CUUAGAUG GGACCTTCTTAGATGC CUGU TGTAAGGTACCTA TMPRSS UGGGACCU  947 CCCTCATGCGTCCTCT 1147 mouse 2_2972 UCUUAGAU GGGACCTTCTTAGATG GCUG CTGTAAGGTACCT TMPRSS UGGAAAGU  948 GACTTAACCTTGAAAT 1148 human 2_2960 CAUGCAAU GGAAAGTCATGCAAT CCCA CCCATTTGCAGGAT TMPRSS CCUUGAAA  949 ACAGCTAGGACTTAA 1149 human 2_2952 UGGAAAGU CCTTGAAATGGAAAG CAUG TCATGCAATCCCATT TMPRSS UACAGCUA  950 TGAAATGAATGATTCT 1150 human 2_2936 GGACUUAA ACAGCTAGGACTTAA CCUU CCTTGAAATGGAAA TMPRSS UUCUACAG  951 GAATGAAATGAATGA 1151 human 2_2933 CUAGGACU TTCTACAGCTAGGACT UAAC TAACCTTGAAATGG TMPRSS UGAAUGAU  952 TTTGCAAGAATGAAAT 1152 human 2_2926 UCUACAGC GAATGATTCTACAGCT UAGG AGGACTTAACCTT TMPRSS GUUUGCAA  953 TGGCCAAGCAGGCTG 1153 human 2_2910 GAAUGAAA GTTTGCAAGAATGAA UGAA ATGAATGATTCTACA TMPRSS CCAAGCAG  954 GGTTCTGCCTCCTGGC 1154 human 2_2898 GCUGGUUU CAAGCAGGCTGGTTTG GCAA CAAGAATGAAATG TMPRSS UGGGUCAU  955 TCAGAAGGCAGTGAA 1155 mouse 2_2877 AACUGGGA TGGGTCATAACTGGG CUCC ACTCCATCTTTGCTG TMPRSS ACCAAAUA  956 AGCCATGCCAGAATT 1156 mouse 2_2796 UGAAGUAU ACCAAATATGAAGTA GAAU TGAATGTCTTACCCA TMPRSS AUUACCAA  957 GAAAGCCATGCCAGA 1157 mouse 2_2793 AUAUGAAG ATTACCAAATATGAA UAUG GTATGAATGTCTTAC TMPRSS UGUGGUCC  958 TTGTTTTGGACTCTCT 1158 human 2_2792 CUUCCAAU GTGGTCCCTTCCAATG GCUG CTGTGGGTTTCCA TMPRSS GCCAGAAU  959 TACCAGGAAAGCCAT 1159 mouse 2_2787 UACCAAAU GCCAGAATTACCAAA AUGA TATGAAGTATGAATG TMPRSS UGCCAGAA  960 ATACCAGGAAAGCCA 1160 mouse 2_2786 UUACCAAA TGCCAGAATTACCAA UAUG ATATGAAGTATGAAT TMPRSS CAUGCCAG  961 TTATACCAGGAAAGC 1161 mouse 2_2784 AAUUACCA CATGCCAGAATTACCA AAUA AATATGAAGTATGA TMPRSS AAAGCCAU  962 TGGGTTTATACCAGGA 1162 mouse 2_2779 GCCAGAAU AAGCCATGCCAGAAT UACC TACCAAATATGAAG TMPRSS UAUACCAG  963 TTCTGGCCCTGGGTTT 1163 mouse 2_2770 GAAAGCCA ATACCAGGAAAGCCA UGCC TGCCAGAATTACCA TMPRSS GAAGAAGA  964 GCCCATGGTGGCGGC 1164 human 2_2757 GAAAGAUG GAAGAAGAGAAAGAT UGUU GTGTTTTGTTTTGGA TMPRSS CGGCGAAG  965 AAGTGCCCATGGTGG 1165 human 2_2753 AAGAGAAA CGGCGAAGAAGAGAA GAUG AGATGTGTTTTGTTT TMPRSS AGAGAGGA  966 TCAGATAAAGATGAA 1166 mouse 2_2727 GAUCAUUG AGAGAGGAGATCATT UCUU GTCTTCTGTCTTCTT TMPRSS UCAGAUAA  967 TAGAAGTTCCTAGCTT 1167 mouse 2_2712 AGAUGAAA CAGATAAAGATGAAA GAGA GAGAGGAGATCATT TMPRSS CCUAGCUU  968 ATGAGATTAGAAGTTC 1168 mouse 2_2705 CAGAUAAA CTAGCTTCAGATAAAG GAUG ATGAAAGAGAGGA TMPRSS CUCCUGAC  969 TGACAAAATGACTGG 1169 human 2_2699 UUAACGUU CTCCTGACTTAACGTT CUAU CTATAAATGAATGT TMPRSS GCUCCUGA  970 TTGACAAAATGACTG 1170 human 2_2698 CUUAACGU GCTCCTGACTTAACGT UCUA TCTATAAATGAATG TMPRSS GGCUCCUG  971 TTTGACAAAATGACTG 1171 human 2_2697 ACUUAACG GCTCCTGACTTAACGT UUCU TCTATAAATGAAT TMPRSS AUUAGAAG  972 TAGACTGGCAATGAG 1172 mouse 2_2695 UUCCUAGC ATTAGAAGTTCCTAGC UUCA TTCAGATAAAGATG TMPRSS AAUGAGAU  973 GTTCTGTAGACTGGCA 1173 mouse 2_2689 UAGAAGUU ATGAGATTAGAAGTTC CCUA CTAGCTTCAGATA TMPRSS GCAAUGAG  974 TTGTTCTGTAGACTGG 1174 mouse 2_2687 AUUAGAAG CAATGAGATTAGAAG UUCC TTCCTAGCTTCAGA TMPRSS UGACAAAA  975 CTATTTCAGCTGCTTT 1175 human 2_2684 UGACUGGC GACAAAATGACTGGC UCCU TCCTGACTTAACGT TMPRSS UUUGACAA  976 GCCTATTTCAGCTGCT 1176 human 2_2682 AAUGACUG TTGACAAAATGACTG GCUC GCTCCTGACTTAAC TMPRSS UAGGUUGU  977 CCTCTTCCAGATGGTT 1177 mouse 2_2668 UCUGUAGA AGGTTGTTCTGTAGAC CUGG TGGCAATGAGATT TMPRSS UUAGGUUG  978 ACCTCTTCCAGATGGT 1178 mouse 2_2667 UUCUGUAG TAGGTTGTTCTGTAGA ACUG CTGGCAATGAGAT TMPRSS UUCCAGAU  979 CAGACACTAGACCTCT 1179 mouse 2_2657 GGUUAGGU TCCAGATGGTTAGGTT UGUU GTTCTGTAGACTG TMPRSS AUCAUCUU  980 ACTCTTTGAAACTGTA 1180 human 2_2641 UGCCAAGU TCATCTTTGCCAAGTA AAGA AGAGTGGTGGCCT TMPRSS UCAGACAC  981 CTGTGTGATTGTGCCT 1181 mouse 2_2641 UAGACCUC CAGACACTAGACCTCT UUCC TCCAGATGGTTAG TMPRSS UGAAACUG  982 CTTCATTTAACTCTTT 1182 human 2_2632 UAUCAUCU GAAACTGTATCATCTT UUGC TGCCAAGTAAGAG TMPRSS UUUGAAAC  983 ACCTTCATTTAACTCT 1183 human 2_2630 UGUAUCAU TTGAAACTGTATCATC CUUU TTTGCCAAGTAAG TMPRSS AACUCUUU  984 AGTCCACCTTCATTTA 1184 human 2_2625 GAAACUGU ACTCTTTGAAACTGTA AUCA TCATCTTTGCCAA TMPRSS UUGGACUG  985 GGAGTTCACCTGCATT 1185 mouse 2_2621 UGUGAUUG TGGACTGTGTGATTGT UGCC GCCTCAGACACTA TMPRSS CCACCUUC  986 ATGTCTCCAAGTAGTC 1186 human 2_2613 AUUUAACU CACCTTCATTTAACTC CUUU TTTGAAACTGTAT TMPRSS UAGUCCAC  987 TTTGATGTCTCCAAGT 1187 human 2_2609 CUUCAUUU AGTCCACCTTCATTTA AACU ACTCTTTGAAACT TMPRSS UUUGAUGU  988 CCTGGAAACTTAGCTT 1188 human 2_2594 CUCCAAGU TTGATGTCTCCAAGTA AGUC GTCCACCTTCATT TMPRSS UUAGCUUU  989 AGTGCTCCTGGAAACT 1189 human 2_2588 UGAUGUCU TAGCTTTTGATGTCTC CCAA CAAGTAGTCCACC TMPRSS CCUGGAAA  990 TGCTACCTCAGTGCTC 1190 human 2_2579 CUUAGCUU CTGGAAACTTAGCTTT UUGA TGATGTCTCCAAG TMPRSS UACUACCU  991 TTGTGTTTCTTCTCTTA 1191 mouse 2_2564 CACUGCAC CTACCTCACTGCACCT CUGG GGACACTAGAGT TMPRSS UCUGGGUU  992 TTCAGTCACCTTGCTT 1192 mouse 2_2543 GUGUUUCU CTGGGTTGTGTTTCTT UCUC CTCTTACTACCTC TMPRSS UUUCCAUG  993 GTTTAAGGTACACTGT 1193 human 2_2540 UUAUGUUU TTCCATGTTATGTTTC CUAC TACACATTGCTAC TMPRSS UACACUGU  994 ATGCTCAGTTTAAGGT 1194 human 2_2533 UUCCAUGU ACACTGTTTCCATGTT UAUG ATGTTTCTACACA TMPRSS GUUUAAGG  995 AATCAAGGATGCTCA 1195 human 2_2525 UACACUGU GTTTAAGGTACACTGT UUCC TTCCATGTTATGTT TMPRSS AGUUUAAG  996 AAATCAAGGATGCTC 1196 human 2_2524 GUACACUG AGTTTAAGGTACACTG UUUC TTTCCATGTTATGT TMPRSS AUCAGAAU  997 TGGAGGCTCAGGTCC 1197 mouse 2_2506 CAGGGACU ATCAGAATCAGGGAC UGUG TTGTGATTTCAGTCA TMPRSS GGGGAAAU  998 AAATTGAGGTCCATG 1198 human 2_2505 CAAGGAUG GGGGAAATCAAGGAT CUCA GCTCAGTTTAAGGTA TMPRSS AGGUCCAU  999 ATCAAATGGAGGCTC 1199 mouse 2_2500 CAGAAUCA AGGTCCATCAGAATC GGGA AGGGACTTGTGATTT TMPRSS UCAGGUCC 1000 TAATCAAATGGAGGC 1200 mouse 2_2498 AUCAGAAU TCAGGTCCATCAGAAT CAGG CAGGGACTTGTGAT TMPRSS UGGAGCCU 1001 TGGCCTAGTACCTGAT 1201 mouse 2_2463 GUAUAGCU GGAGCCTGTATAGCTC CAGC AGCTAATCAAATG TMPRSS ACAGGCAU 1002 CTCTTAGCTTTGGCTA 1202 mouse 2_2441 GGCCUAGU CAGGCATGGCCTAGT ACCU ACCTGATGGAGCCT TMPRSS CCUGAAAC 1003 CTCAGCCTCTCAGAGC 1203 mouse 2_2414 UUACCUCU CTGAAACTTACCTCTT UAGC AGCTTTGGCTACA TMPRSS UCUCAGAG 1004 CTGTTTCTCTCAGCCT 1204 mouse 2_2406 CCUGAAAC CTCAGAGCCTGAAACT UUAC TACCTCTTAGCTT TMPRSS UGGGUGAG 1005 TGCCGGCATGTCCCTT 1205 mouse 2_2341 CUCUACAU GGGTGAGCTCTACATG GGUG GTGTTATTCAGTC TMPRSS AGAGCAAG 1006 CTAGGCAGATCTCTCA 1206 mouse 2_2311 AAGCUAAU GAGCAAGAAGCTAAT GCCG GCCGGCATGTCCCT TMPRSS UUCCCAAG 1007 AGGGTGATGGAGGCT 1207 mouse 2_2280 CUAAGGGC TTCCCAAGCTAAGGGC CUAG CTAGGCAGATCTCT TMPRSS AUAGACAG 1008 TTATGGGGTGAGAAT 1208 human 2_2257 UGCCCUUG ATAGACAGTGCCCTTG GUGC GTGCGAGGGAAGCA TMPRSS GGUGACGU 1009 CAGCCCTTCATGGGTG 1209 human 2_2196 GGUAGUCA GTGACGTGGTAGTCAC CUUG TTGTAAGGGGAAC TMPRSS UGGGCUGG 1010 ATCTGCTGTGCAGGTT 1210 mouse 2_2165 UCAUACUG GGGCTGGTCATACTGT UCAU CATGATTTCATTA TMPRSS GUUGGGCU 1011 AAATCTGCTGTGCAGG 1211 mouse 2_2163 GGUCAUAC TTGGGCTGGTCATACT UGUC GTCATGATTTCAT TMPRSS UGAGUAAC 1012 AAGGACTATGACCTCT 1212 mouse 2_2047 CUGAUGAC GAGTAACCTGATGAC CUGA CTGAGAAAGAGTAA TMPRSS GACCUCUG 1013 ATCACTAAGGACTATG 1213 mouse 2_2041 AGUAACCU ACCTCTGAGTAACCTG GAUG ATGACCTGAGAAA TMPRSS UGGAUCAC 1014 TGCTCAGGCCTTTTTT 1214 mouse 2_2023 UAAGGACU GGATCACTAAGGACT AUGA ATGACCTCTGAGTA TMPRSS UUGGAUCA 1015 ATGCTCAGGCCTTTTT 1215 mouse 2_2022 CUAAGGAC TGGATCACTAAGGACT UAUG ATGACCTCTGAGT TMPRSS ACUCUCAU 1016 GCAGAGGAGGGTGGC 1216 mouse 2_1978 GUUGGAAC ACTCTCATGTTGGAAC UUCU TTCTTTTGGGCTCA TMPRSS CUUUCCAG 1017 TCTTCCTGCTGAGTCC 1217 human 2_1952 GGGCCAAU TTTCCAGGGGCCAATT UUUG TTGGATGAGCATG TMPRSS ACCACCAG 1018 TTTGAACTCAGGGTCA 1218 human 2_193 CUAUUGGA CCACCAGCTATTGGAC CCUU CTTACTATGAAAA TMPRSS UGCCCCAU 1019 GAGAGGGGTGGAGGC 1219 human 2_1924 UGAGAUCU TGCCCCATTGAGATCT UCCU TCCTGCTGAGTCCT TMPRSS GAUGACCA 1020 GCTCCCACCAGAATG 1220 mouse 2_1922 GAUUCUGU GATGACCAGATTCTGT UGGG TGGGTTTGGGCACA TMPRSS CCCACCAG 1021 GCCACTTCTGCAGCTC 1221 mouse 2_1910 AAUGGAUG CCACCAGAATGGATG ACCA ACCAGATTCTGTTG TMPRSS UUCACUUU 1022 TGATTCGAAGGGCCTT 1222 mouse 2_1821 UAUUAAAC TCACTTTTATTAAACA AGUG GTGACTTGTTTGA TMPRSS UUUCACUU 1023 ATGATTCGAAGGGCCT 1223 mouse 2_1820 UUAUUAAA TTCACTTTTATTAAAC CAGU AGTGACTTGTTTG TMPRSS CGAAGGGC 1024 CCTTTTGAGATGATTC 1224 mouse 2_1811 CUUUCACU GAAGGGCCTTTCACTT UUUA TTATTAAACAGTG TMPRSS UUUGAGAU 1025 GTGCACAATGTACCTT 1225 mouse 2_1799 GAUUCGAA TTGAGATGATTCGAAG GGGC GGCCTTTCACTTT TMPRSS UGUACCUU 1026 ACTTCCTGTGCACAAT 1226 mouse 2_1792 UUGAGAUG GTACCTTTTGAGATGA AUUC TTCGAAGGGCCTT TMPRSS GUGCACAA 1027 GAATTTTAACTTCCTG 1227 mouse 2_1784 UGUACCUU TGCACAATGTACCTTT UUGA TGAGATGATTCGA TMPRSS CUGUGCAC 1028 CTGAATTTTAACTTCC 1228 mouse 2_1782 AAUGUACC TGTGCACAATGTACCT UUUU TTTGAGATGATTC TMPRSS CCUGAAUU 1029 CAAGAAAACCAAGGG 1229 mouse 2_1766 UUAACUUC CCTGAATTTTAACTTC CUGU CTGTGCACAATGTA TMPRSS UAAACAGU 1030 TCACTTCATTTTTATT 1230 human 2_1764 GAACUUGU AAACAGTGAACTTGTC CUGG TGGCTTTGGCACT TMPRSS UUAAACAG 1031 GTCACTTCATTTTTAT 1231 human 2_1763 UGAACUUG TAAACAGTGAACTTGT UCUG CTGGCTTTGGCAC TMPRSS AGGGCCUG 1032 TCTGCAAGAAAACCA 1232 mouse 2_1762 AAUUUUAA AGGGCCTGAATTTTAA CUUC CTTCCTGTGCACAA TMPRSS AAGGGCCU 1033 TTCTGCAAGAAAACC 1233 mouse 2_1761 GAAUUUUA AAGGGCCTGAATTTTA ACUU ACTTCCTGTGCACA TMPRSS CAAGAAAA 1034 TTCAACAACCTTCTGC 1234 mouse 2_1751 CCAAGGGC AAGAAAACCAAGGGC CUGA CTGAATTTTAACTT TMPRSS UGCAAGAA 1035 TCTTCAACAACCTTCT 1235 mouse 2_1749 AACCAAGG GCAAGAAAACCAAGG GCCU GCCTGAATTTTAAC TMPRSS CUGCAAGA 1036 GTCTTCAACAACCTTC 1236 mouse 2_1748 AAACCAAG TGCAAGAAAACCAAG GGCC GGCCTGAATTTTAA TMPRSS UUCUGCAA 1037 TTGTCTTCAACAACCT 1237 mouse 2_1746 GAAAACCA TCTGCAAGAAAACCA AGGG AGGGCCTGAATTTT TMPRSS AUUCAGAG 1038 TTACTCTTAGAGATGA 1238 human 2_1740 GUCACUUC TTCAGAGGTCACTTCA AUUU TTTTTATTAAACA TMPRSS UGAUUCAG 1039 ATTTACTCTTAGAGAT 1239 human 2_1738 AGGUCACU GATTCAGAGGTCACTT UCAU CATTTTTATTAAA TMPRSS UUAGAGAU 1040 GTGCATGATTTACTCT 1240 human 2_1731 GAUUCAGA TAGAGATGATTCAGA GGUC GGTCACTTCATTTT TMPRSS UUUACUCU 1041 CTTCCCCGTGCATGAT 1241 human 2_1724 UAGAGAUG TTACTCTTAGAGATGA AUUC TTCAGAGGTCACT TMPRSS UGUCCCAG 1042 TAATCCACGTGGCTTT 1242 mouse 2_1716 ACUUCCUU GTCCCAGACTTCCTTT UGUC GTCTTCAACAACC TMPRSS CCGUGCAU 1043 GCTGGTTTTGCTTCCC 1243 human 2_1714 GAUUUACU CGTGCATGATTTACTC CUUA TTAGAGATGATTC TMPRSS GGCUUUGU 1044 ACAGCTAATCCACGTG 1244 mouse 2_1711 CCCAGACU GCTTTGTCCCAGACTT UCCU CCTTTGTCTTCAA TMPRSS UUCCCCGU 1045 TGGGGCTGGTTTTGCT 1245 human 2_1710 GCAUGAUU TCCCCGTGCATGATTT UACU ACTCTTAGAGATG TMPRSS UUACAAGA 1046 GTCCTTGACGTCGTTT 1246 human 2_1681 AAACAAUG TACAAGAAAACAATG GGGC GGGCTGGTTTTGCT TMPRSS UUUACAAG 1047 CGTCCTTGACGTCGTT 1247 human 2_1680 AAAACAAU TTACAAGAAAACAAT GGGG GGGGCTGGTTTTGC TMPRSS UUUUACAA 1048 TCGTCCTTGACGTCGT 1248 human 2_1679 GAAAACAA TTTACAAGAAAACAA UGGG TGGGGCTGGTTTTG TMPRSS UUCGUCCU 1049 CTAATCCACATGGTCT 1249 human 2_1663 UGACGUCG TCGTCCTTGACGTCGT UUUU TTTACAAGAAAAC TMPRSS ACGGUAUU 1050 GTATACGGGAACGTG 1250 mouse 2_1656 UACAGAUU ACGGTATTTACAGATT GGAU GGATCTACCAGCAA TMPRSS GACGGUAU 1051 AGTATACGGGAACGT 1251 mouse 2_1655 UUACAGAU GACGGTATTTACAGAT UGGA TGGATCTACCAGCA TMPRSS ACGGGAAC 1052 TCAGACCTGGAGTATA 1252 mouse 2_1645 GUGACGGU CGGGAACGTGACGGT AUUU ATTTACAGATTGGA TMPRSS AUGGUAUU 1053 GTGTACGGGAATGTG 1253 human 2_1604 CACGGACU ATGGTATTCACGGACT GGAU GGATTTATCGACAA TMPRSS GAUGGUAU 1054 AGTGTACGGGAATGT 1254 human 2_1603 UCACGGAC GATGGTATTCACGGAC UGGA TGGATTTATCGACA TMPRSS GGGAAUGU 1055 AGACCAGGAGTGTAC 1255 human 2_1595 GAUGGUAU GGGAATGTGATGGTA UCAC TTCACGGACTGGATT TMPRSS ACGGGAAU 1056 ACAGACCAGGAGTGT 1256 human 2_1593 GUGAUGGU ACGGGAATGTGATGG AUUC TATTCACGGACTGGA TMPRSS GUUGAUAA 1057 TCATTACTCGATGCTG 1257 human 2_158 CAGCAAGA TTGATAACAGCAAGA UGGC TGGCTTTGAACTCA TMPRSS CUGUUGAU 1058 TATCATTACTCGATGC 1258 human 2_156 AACAGCAA TGTTGATAACAGCAA GAUG GATGGCTTTGAACT TMPRSS UCUGGCUG 1059 GATACAAGCTGGGGT 1259 human 2_1559 UGCCAAAG TCTGGCTGTGCCAAAG CUUA CTTACAGACCAGGA TMPRSS GGUUACUU 1060 CAGTGGAGGGCCGCT 1260 mouse 2_1559 UGAAGAAU GGTTACTTTGAAGAAT GGGA GGGATCTGGTGGCT TMPRSS UGUAAUAG 1061 ATCGAGCCCTCCAAAT 1261 mouse 2_1452 UAAAUACA GTAATAGTAAATACAT UAUA ATACAACAACCTA TMPRSS CCAAAUGU 1062 CCTTGATCGAGCCCTC 1262 mouse 2_1447 AAUAGUAA CAAATGTAATAGTAA AUAC ATACATATACAACA TMPRSS CUCCAAAU 1063 ACCCTTGATCGAGCCC 1263 mouse 2_1445 GUAAUAGU TCCAAATGTAATAGTA AAAU AATACATATACAA TMPRSS CGAGCCCU 1064 CATGGTACCCTTGATC 1264 mouse 2_1439 CCAAAUGU GAGCCCTCCAAATGTA AAUA ATAGTAAATACAT TMPRSS GAUCGAGC 1065 TGCCATGGTACCCTTG 1265 mouse 2_1436 CCUCCAAA ATCGAGCCCTCCAAAT UGUA GTAATAGTAAATA TMPRSS AUGUCUAU 1066 GATGCAACAGCAGAT 1266 human 2_1413 GACAACCU ATGTCTATGACAACCT GAUC GATCACACCAGCCA TMPRSS UCGGACGU 1067 GAGAAAGGGAAGACC 1267 mouse 2_1404 GUUGAAUG TCGGACGTGTTGAATG CUGC CTGCCATGGTACCC TMPRSS AACAGCAG 1068 GAGACACAGAGATGC 1268 human 2_1403 AUAUGUCU AACAGCAGATATGTCT AUGA ATGACAACCTGATC TMPRSS CUCGGACG 1069 TGAGAAAGGGAAGAC 1269 mouse 2_1403 UGUUGAAU CTCGGACGTGTTGAAT GCUG GCTGCCATGGTACC TMPRSS AGAUGCAA 1070 CTCATTGAGACACAG 1270 human 2_1397 CAGCAGAU AGATGCAACAGCAGA AUGU TATGTCTATGACAAC TMPRSS UGAGAAAG 1071 GTGGGGGGCCACCTA 1271 mouse 2_1388 GGAAGACC TGAGAAAGGGAAGAC UCGG CTCGGACGTGTTGAA TMPRSS AUACCUAU 1072 TATTGAACATTCCAGA 1272 human 2_136 CAUUACUC TACCTATCATTACTCG GAUG ATGCTGTTGATAA TMPRSS UCAGAAGU 1073 GAGAAAGGGAAGACC 1273 human 2_1352 GCUGAACG TCAGAAGTGCTGAAC CUGC GCTGCCAAGGTGCTT TMPRSS CUCAGAAG 1074 GGAGAAAGGGAAGAC 1274 human 2_1351 UGCUGAAC CTCAGAAGTGCTGAA GCUG CGCTGCCAAGGTGCT TMPRSS CCAGAUAC 1075 GTCATATTGAACATTC 1275 human 2_132 CUAUCAUU CAGATACCTATCATTA ACUC CTCGATGCTGTTG TMPRSS UCCAGAUA 1076 GGTCATATTGAACATT 1276 human 2_131 CCUAUCAU CCAGATACCTATCATT UACU ACTCGATGCTGTT TMPRSS UUGGCUUU 1077 AAGCTGCAGACACCTT 1277 mouse 2_1287 UAAUGAUC TGGCTTTTAATGATCT UAGU AGTGAAGCCAGTG TMPRSS UGAAGCUG 1078 ACGACATTGCTCTCAT 1278 mouse 2_1270 CAGACACC GAAGCTGCAGACACC UUUG TTTGGCTTTTAATG TMPRSS AACGACAU 1079 TCTAAGACCAAGAAT 1279 mouse 2_1254 UGCUCUCA AACGACATTGCTCTCA UGAA TGAAGCTGCAGACA TMPRSS GCAGAAGC 1080 TGCGCTGATGAAGCTG 1280 human 2_1225 CUCUGACU CAGAAGCCTCTGACTT UUCA TCAACGACCTAGT TMPRSS UUCCCAUC 1081 GGTAGAAAAAGTAAT 1281 mouse 2_1220 CAAAUUAC TTCCCATCCAAATTAC GACU GACTCTAAGACCAA TMPRSS GGAAGUAG 1082 TCTCTCATGTTCTATG 1282 mouse 2_1191 ACACCAGG GAAGTAGACACCAGG UAGA TAGAAAAAGTAATT TMPRSS UGGAAGUA 1083 GTCTCTCATGTTCTAT 1283 mouse 2_1190 GACACCAG GGAAGTAGACACCAG GUAG GTAGAAAAAGTAAT TMPRSS UGUUCUAU 1084 TGAGACAGTCTCTCAT 1284 mouse 2_1183 GGAAGUAG GTTCTATGGAAGTAGA ACAC CACCAGGTAGAAA TMPRSS UAUGACUC 1085 ATTTCTCATCCAAATT 1285 human 2_1181 CAAGACCA ATGACTCCAAGACCA AGAA AGAACAATGACATT TMPRSS UUCUCAUC 1086 AGTAGAAAAAGTGAT 1286 human 2_1168 CAAAUUAU TTCTCATCCAAATTAT GACU GACTCCAAGACCAA TMPRSS UGAGACAG 1087 CATTTGCGGGAATTCT 1287 mouse 2_1168 UCUCUCAU GAGACAGTCTCTCATG GUUC TTCTATGGAAGTA TMPRSS CUGAGACA 1088 GCATTTGCGGGAATTC 1288 mouse 2_1167 GUCUCUCA TGAGACAGTCTCTCAT UGUU GTTCTATGGAAGT TMPRSS UGCGGGAA 1089 GTACTGGACGGCATTT 1289 mouse 2_1157 UUCUGAGA GCGGGAATTCTGAGA CAGU CAGTCTCTCATGTT TMPRSS CAGGUCAU 1090 CCGCCTGGAGCGCGG 1290 human 2_114 AUUGAACA CAGGTCATATTGAACA UUCC TTCCAGATACCTAT TMPRSS GAGACAAU 1091 ATTTGCGGGGATTTTG 1291 human 2_1117 CUUUCAUG AGACAATCTTTCATGT UUCU TCTATGGAGCCGG TMPRSS CGCGGCAG 1092 GAGCGCCGCCTGGAG 1292 human 2_109 GUCAUAUU CGCGGCAGGTCATATT GAAC GAACATTCCAGATA TMPRSS AUGGCAUU 1093 ACCTCTTAACAATCCA 1293 human 2_1087 GGACGGCA TGGCATTGGACGGCAT UUUG TTGCGGGGATTTT TMPRSS CAUGGCAU 1094 AACCTCTTAACAATCC 1294 human 2_1086 UGGACGGC ATGGCATTGGACGGC AUUU ATTTGCGGGGATTT

TABLE 11B Host target IL-6 - 20 nucleotide targets and 45 nucleotide gene target regions SEQ SEQ SEQUENCE ID ID ID 20 nt Sequence NO: 45 nt Gene Region NO: Species IL6_999 UUGUAUUU 1295 AAAGAAATATTTATATTG 1495 human AUAUAAUG TATTTATATAATGTATAAA UAUA TGGTTTTT IL6_998 AUUGUAUU 1296 TAAAGAAATATTTATATT 1496 human UAUAUAAU GTATTTATATAATGTATAA GUAU ATGGTTTT IL6_995 UAUAUUGU 1297 TTTTAAAGAAATATTTATA 1497 human AUUUAUAU TTGTATTTATATAATGTAT AAUG AAATGGT IL6_994 UUAUAUUG 1298 TTTTTAAAGAAATATTTAT 1498 human UAUUUAUA ATTGTATTTATATAATGTA UAAU TAAATGG IL6_989 AAUAUUUA 1299 ACATATTTTTAAAGAAAT 1499 human UAUUGUAU ATTTATATTGTATTTATAT UUAU AATGTATA IL6_984 AAAGAAAU 1300 CTTATACATATTTTTAAAG 1500 human AUUUAUAU AAATATTTATATTGTATTT UGUA ATATAAT IL6_982 UUAAAGAA 1301 AACTTATACATATTTTTAA 1501 human AUAUUUAU AGAAATATTTATATTGTAT AUUG TTATATA IL6_980 UUUUAAAG 1302 CTAACTTATACATATTTTT 1502 human AAAUAUUU AAAGAAATATTTATATTG AUAU TATTTATA IL6_963 GGCUAACUU 1303 TTACCTCAAATAAATGGC 1503 human AUACAUAUU TAACTTATACATATTTTTA UU AAGAAATA IL6_962 UGGCUAACU 1304 CTTACCTCAAATAAATGG 1504 human UAUACAUAU CTAACTTATACATATTTTT UU AAAGAAAT IL6_955 AAAUAAAU 1305 GTGTAGGCTTACCTCAAA 1505 human GGCUAACUU TAAATGGCTAACTTATAC AUA ATATTTTTA IL6_951 CCUCAAAUA 1306 GAAAGTGTAGGCTTACCT 1506 human AAUGGCUAA CAAATAAATGGCTAACTT CU ATACATATT IL6_950 ACCUCAAAU 1307 GGAAAGTGTAGGCTTACC 1507 human AAAUGGCUA TCAAATAAATGGCTAACT AC TATACATAT IL6_949 UACCUCAAA 1308 TGGAAAGTGTAGGCTTAC 1508 human UAAAUGGCU CTCAAATAAATGGCTAAC AA TTATACATA IL6_948 UUACCUCAA 1309 TTGGAAAGTGTAGGCTTA 1509 human AUAAAUGGC CCTCAAATAAATGGCTAA UA CTTATACAT IL6_947 CUUACCUCA 1310 CTTGGAAAGTGTAGGCTT 1510 human AAUAAAUG ACCTCAAATAAATGGCTA GCU ACTTATACA IL6_943 UAGGCUUAC 1311 ATTTCTTGGAAAGTGTAG 1511 human CUCAAAUAA GCTTACCTCAAATAAATG AU GCTAACTTA IL6_934 UGGAAAGU 1312 AGCCAGATCATTTCTTGG 1512 human GUAGGCUUA AAAGTGTAGGCTTACCTC CCU AAATAAATG IL6_932 CUUGGAAAG 1313 AGAGCCAGATCATTTCTT 1513 human UGUAGGCUU GGAAAGTGTAGGCTTACC AC TCAAATAAA IL6_917 AGAGCCAGA 1314 AATATCCTTTGTTTCAGAG 1514 human UCAUUUCUU CCAGATCATTTCTTGGAA GG AGTGTAGG IL6_912 GUUUCAGAG 1315 GTTTGAATATCCTTTGTTT 1515 human CCAGAUCAU CAGAGCCAGATCATTTCTT UU GGAAAGT IL6_894 GCAGUUUGA 1316 ATGGAAAGTGGCTATGCA 1516 human AUAUCCUUU GTTTGAATATCCTTTGTTT GU CAGAGCCA IL6_878 AAUGGAAA 1317 TAGTTTTGAAATAATAAT 1517 human GUGGCUAUG GGAAAGTGGCTATGCAGT CAG TTGAATATC IL6_870 GAAAUAAU 1318 TTATGTATTAGTTTTGAAA 1518 human AAUGGAAA TAATAATGGAAAGTGGCT GUGG ATGCAGTT IL6_867 UUUGAAAU 1319 ATTTTATGTATTAGTTTTG 1519 human AAUAAUGG AAATAATAATGGAAAGTG AAAG GCTATGCA IL6_858 UGUAUUAG 1320 ACTTGAAACATTTTATGTA 1520 human UUUUGAAA TTAGTTTTGAAATAATAAT UAAU GGAAAGT IL6_856 UAUGUAUU 1321 CCACTTGAAACATTTTATG 1521 human AGUUUUGA TATTAGTTTTGAAATAATA AAUA ATGGAAA IL6_851 CAUUUUAUG 1322 AAGTACCACTTGAAACAT 1522 human UAUUAGUU TTTATGTATTAGTTTTGAA UUG ATAATAAT IL6_843 ACUUGAAAC 1323 TTTTTAAGAAGTACCACTT 1523 human AUUUUAUG GAAACATTTTATGTATTAG UAU TTTTGAA IL6_840 ACCACUUGA 1324 ATATTTTTAAGAAGTACC 1524 human AACAUUUUA ACTTGAAACATTTTATGTA UG TTAGTTTT IL6_839 UACCACUUG 1325 TATATTTTTAAGAAGTACC 1525 human AAACAUUUU ACTTGAAACATTTTATGTA AU TTAGTTT IL6_838 GUACCACUU 1326 TTATATTTTTAAGAAGTAC 1526 human GAAACAUUU CACTTGAAACATTTTATGT UA ATTAGTT IL6_831 UUAAGAAG 1327 GTCATATTTATATTTTTAA 1527 human UACCACUUG GAAGTACCACTTGAAACA AAA TTTTATGT IL6_830 UUUAAGAA 1328 AGTCATATTTATATTTTTA 1528 human GUACCACUU AGAAGTACCACTTGAAAC GAA ATTTTATG IL6_829 UUUUAAGA 1329 AAGTCATATTTATATTTTT 1529 human AGUACCACU AAGAAGTACCACTTGAAA UGA CATTTTAT IL6_809 UAUGUAAG 1330 GAAGCTGAGTTAATTTAT 1530 human UCAUAUUUA GTAAGTCATATTTATATTT UAU TTAAGAAG IL6_808 UUAUGUAA 1331 TGAAGCTGAGTTAATTTAT 1531 human GUCAUAUUU GTAAGTCATATTTATATTT AUA TTAAGAA IL6_805 AAUUUAUG 1332 ATGTGAAGCTGAGTTAAT 1532 human UAAGUCAUA TTATGTAAGTCATATTTAT UUU ATTTTTAA IL6_804 UAAUUUAU 1333 TATGTGAAGCTGAGTTAA 1533 human GUAAGUCAU TTTATGTAAGTCATATTTA AUU TATTTTTA IL6_803 UUAAUUUA 1334 ATATGTGAAGCTGAGTTA 1534 human UGUAAGUCA ATTTATGTAAGTCATATTT UAU ATATTTTT IL6_801 AGUUAAUU 1335 AAATATGTGAAGCTGAGT 1535 human UAUGUAAG TAATTTATGTAAGTCATAT UCAU TTATATTT IL6_800 GAGUUAAU 1336 TAAATATGTGAAGCTGAG 1536 human UUAUGUAA TTAATTTATGTAAGTCATA GUCA TTTATATT IL6_799 UGAGUUAA 1337 TTAAATATGTGAAGCTGA 1537 human UUUAUGUA GTTAATTTATGTAAGTCAT AGUC ATTTATAT IL6_798 CUGAGUUAA 1338 TTTAAATATGTGAAGCTG 1538 human UUUAUGUA AGTTAATTTATGTAAGTCA AGU TATTTATA IL6_794 GAAGCUGAG 1339 AATATTTAAATATGTGAA 1539 human UUAAUUUA GCTGAGTTAATTTATGTAA UGU GTCATATT IL6_793 UGAAGCUGA 1340 TAATATTTAAATATGTGA 1540 human GUUAAUUU AGCTGAGTTAATTTATGTA AUG AGTCATAT IL6_792 GUGAAGCUG 1341 TTAATATTTAAATATGTGA 1541 human AGUUAAUU AGCTGAGTTAATTTATGTA UAU AGTCATA IL6_790 AUGUGAAGC 1342 TATTAATATTTAAATATGT 1542 human UGAGUUAA GAAGCTGAGTTAATTTAT UUU GTAAGTCA IL6_788 AUAUGUGA 1343 TTTATTAATATTTAAATAT 1543 human AGCUGAGUU GTGAAGCTGAGTTAATTT AAU ATGTAAGT IL6_784 UUAAAUAU 1344 TTAATTTATTAATATTTAA 1544 human GUGAAGCUG ATATGTGAAGCTGAGTTA AGU ATTTATGT IL6_783 UUUAAAUA 1345 TTTAATTTATTAATATTTA 1545 human UGUGAAGCU AATATGTGAAGCTGAGTT GAG AATTTATG IL6_782 AUUUAAAU 1346 TTTTAATTTATTAATATTT 1546 human AUGUGAAGC AAATATGTGAAGCTGAGT UGA TAATTTAT IL6_778 UAAUAUUU 1347 TTATTTTTAATTTATTAAT 1547 human AAAUAUGU ATTTAAATATGTGAAGCT GAAG GAGTTAAT IL6_776 AUUAAUAU 1348 AATTATTTTTAATTTATTA 1548 human UUAAAUAU ATATTTAAATATGTGAAG GUGA CTGAGTTA IL6_774 UUAUUAAU 1349 TTAATTATTTTTAATTTAT 1549 human AUUUAAAU TAATATTTAAATATGTGA AUGU AGCTGAGT IL6_770 UAAUUUAU 1350 TATTTTAATTATTTTTAAT 1550 human UAAUAUUU TTATTAATATTTAAATATG AAAU TGAAGCT IL6_768 UUUAAUUU 1351 ACTATTTTAATTATTTTTA 1551 human AUUAAUAU ATTTATTAATATTTAAATA UUAA TGTGAAG IL6_747 UAGGACACU 1352 TAAAAGTATGAGCGTTAG 1552 human AUUUUAAU GACACTATTTTAATTATTT UAU TTAATTTA IL6_746 UUAGGACAC 1353 CTAAAAGTATGAGCGTTA 1553 human UAUUUUAA GGACACTATTTTAATTATT UUA TTTAATTT IL6_745 GUUAGGACA 1354 ACTAAAAGTATGAGCGTT 1554 human CUAUUUUAA AGGACACTATTTTAATTAT UU TTTTAATT IL6_743 GCGUUAGGA 1355 GAACTAAAAGTATGAGCG 1555 human CACUAUUUU TTAGGACACTATTTTAATT AA ATTTTTAA IL6_742 AGCGUUAGG 1356 AGAACTAAAAGTATGAGC 1556 human ACACUAUUU GTTAGGACACTATTTTAAT UA TATTTTTA IL6_741 GAGCGUUAG 1357 GAGAACTAAAAGTATGAG 1557 human GACACUAUU CGTTAGGACACTATTTTAA UU TTATTTTT IL6_740 UGAGCGUUA 1358 GGAGAACTAAAAGTATGA 1558 human GGACACUAU GCGTTAGGACACTATTTTA UU ATTATTTT IL6_739 AUGAGCGUU 1359 TGGAGAACTAAAAGTATG 1559 human AGGACACUA AGCGTTAGGACACTATTTT UU AATTATTT IL6_731 CUAAAAGUA 1360 GTTCTCTATGGAGAACTA 1560 human UGAGCGUUA AAAGTATGAGCGTTAGGA GG CACTATTTT IL6_723 AUGGAGAAC 1361 CTTATGTTGTTCTCTATGG 1561 human UAAAAGUA AGAACTAAAAGTATGAGC UGA GTTAGGAC IL6_717 UUCUCUAUG 1362 ACAGAACTTATGTTGTTCT 1562 human GAGAACUAA CTATGGAGAACTAAAAGT AA ATGAGCGT IL6_697 UGGGCACAG 1363 CAGAAACCTGTCCACTGG 1563 human AACUUAUGU GCACAGAACTTATGTTGTT UG CTCTATGG IL6_691 GUCCACUGG 1364 TCTGGTCAGAAACCTGTC 1564 human GCACAGAAC CACTGGGCACAGAACTTA UU TGTTGTTCT IL6_654 GUUGUUAA 1365 GCACCTCAGATTGTTGTTG 1565 human UGGGCAUUC TTAATGGGCATTCCTTCTT CUU CTGGTCA IL6_629 UGUAGCAUG 1366 GGGCTCTTCGGCAAATGT 1566 human GGCACCUCA AGCATGGGCACCTCAGAT GA TGTTGTTGT IL6_580 CGCAGCUUU 1367 ACTCATCTCATTCTGCGCA 1567 human AAGGAGUUC GCTTTAAGGAGTTCCTGC CU AGTCCAGC IL6_58 CCAGCUAUG 1368 CTCCCCTCCAGGAGCCCA 1568 human AACUCCUUC GCTATGAACTCCTTCTCCA UC CAATACCC IL6_561 GACAACUCA 1369 GTGGCTGCAGGACATGAC 1569 human UCUCAUUCU AACTCATCTCATTCTGCGC GC AGCTTTAA IL6_54 GAGCCCAGC 1370 CTATCTCCCCTCCAGGAGC 1570 human UAUGAACUC CCAGCTATGAACTCCTTCT CU CCACAAT IL6_427 AUGAGUACA 1371 GCCAGAGCTGTGCAGATG 1571 human AAAGUCCUG AGTACAAAAGTCCTGATC AU CAGTTCCTG IL6_423 GCAGAUGAG 1372 ACAAGCCAGAGCTGTGCA 1572 human UACAAAAGU GATGAGTACAAAAGTCCT CC GATCCAGTT IL6_404 AGGAACAAG 1373 GATTTGAGAGTAGTGAGG 1573 human CCAGAGCUG AACAAGCCAGAGCTGTGC UG AGATGAGTA IL6_382 CAGAACAGA 1374 TACCTAGAGTACCTCCAG 1574 human UUUGAGAG AACAGATTTGAGAGTAGT UAG GAGGAACAA IL6_381 CCAGAACAG 1375 ATACCTAGAGTACCTCCA 1575 human AUUUGAGA GAACAGATTTGAGAGTAG GUA TGAGGAACA IL6_352 UUGGAGUU 1376 ATCATCACTGGTCTTTTGG 1576 human UGAGGUAU AGTTTGAGGTATACCTAG ACCU AGTACCTC IL6_343 ACUGGUCUU 1377 CTGGTGAAAATCATCACT 1577 human UUGGAGUU GGTCTTTTGGAGTTTGAGG UGA TATACCTA IL6_339 CAUCACUGG 1378 TTGCCTGGTGAAAATCAT 1578 human UCUUUUGGA CACTGGTCTTTTGGAGTTT GU GAGGTATA IL6_331 GUGAAAAUC 1379 GAGGAGACTTGCCTGGTG 1579 human AUCACUGGU AAAATCATCACTGGTCTTT CU TGGAGTTT IL6_325 UGCCUGGUG 1380 TTCAATGAGGAGACTTGC 1580 human AAAAUCAUC CTGGTGAAAATCATCACT AC GGTCTTTTG IL6_324 UUGCCUGGU 1381 ATTCAATGAGGAGACTTG 1581 human GAAAAUCAU CCTGGTGAAAATCATCAC CA TGGTCTTTT IL6_311 UCAAUGAGG 1382 GCTTCCAATCTGGATTCAA 1582 human AGACUUGCC TGAGGAGACTTGCCTGGT UG GAAAATCA IL6_309 AUUCAAUGA 1383 ATGCTTCCAATCTGGATTC 1583 human GGAGACUUG AATGAGGAGACTTGCCTG CC GTGAAAAT IL6_296 GCUUCCAAU 1384 CTGAAAAAGATGGATGCT 1584 human CUGGAUUCA TCCAATCTGGATTCAATG AU AGGAGACTT IL6_248 UGGCAGAAA 1385 GCAGCAAAGAGGCACTGG 1585 human ACAACCUGA CAGAAAACAACCTGAACC AC TTCCAAAGA IL6_217 AGUAACAUG 1386 GAGACATGTAACAAGAGT 1586 human UGUGAAAGC AACATGTGTGAAAGCAGC AG AAAGAGGCA IL6_210 UAACAAGAG 1387 GAGAAAGGAGACATGTAA 1587 human UAACAUGUG CAAGAGTAACATGTGTGA UG AAGCAGCAA IL6_209 GUAACAAGA 1388 TGAGAAAGGAGACATGTA 1588 human GUAACAUGU ACAAGAGTAACATGTGTG GU AAAGCAGCA IL6_203 AGACAUGUA 1389 CAGCCCTGAGAAAGGAGA 1589 human ACAAGAGUA CATGTAACAAGAGTAACA AC TGTGTGAAA IL6_201 GGAGACAUG 1390 CTCAGCCCTGAGAAAGGA 1590 human UAACAAGAG GACATGTAACAAGAGTAA UA CATGTGTGA IL6_1008 UAUAAUGU 1391 TTTATATTGTATTTATATA 1591 human AUAAAUGG ATGTATAAATGGTTTTTAT UUUU ACCAATA IL6_1007 AUAUAAUG 1392 ATTTATATTGTATTTATAT 1592 human UAUAAAUG AATGTATAAATGGTTTTTA GUUU TACCAAT IL6_1006 UAUAUAAU 1393 TATTTATATTGTATTTATA 1593 human GUAUAAAU TAATGTATAAATGGTTTTT GGUU ATACCAA IL6_1000 UGUAUUUA 1394 AAGAAATATTTATATTGT 1594 human UAUAAUGU ATTTATATAATGTATAAAT AUAA GGTTTTTA IL6_937 UGUUAUAU 1395 AAGTGTCACTTGAAATGT 1595 mouse GUUAUAGU TATATGTTATAGTTTTGAA UUUG ATGATAAC IL6_402 UUGCCUAUU 1396 ATATAATCAGGAAATTTG 1596 mouse GAAAAUUUC CCTATTGAAAATTTCCTCT CU GGTCTTCT IL6_1044 UUGCUAAUU 1397 GTTTACCTCAATGAATTGC 1597 mouse UAAAUAUG TAATTTAAATATGTTTTTA UUU AAGAAAT IL6_891 UAAUUUAU 1398 AAGTAAACTTTAAGTTAA 1598 mouse GAUUGAUA TTTATGATTGATATTTATT UUUA ATTTTTAT IL6_855 UAAUUUAU 1399 TATTTTAATTATTTTTAAT 1599 mouse UGAUAAUU TTATTGATAATTTAAATAA UAAA GTAAACT IL6_1036 UCAAUGAAU 1400 ATGTATAAGTTTACCTCAA 1600 mouse UGCUAAUUU TGAATTGCTAATTTAAATA AA TGTTTTT IL6_886 UAAGUUAA 1401 TAAATAAGTAAACTTTAA 1601 mouse UUUAUGAU GTTAATTTATGATTGATAT UGAU TTATTATT IL6_884 UUUAAGUU 1402 TTTAAATAAGTAAACTTTA 1602 mouse AAUUUAUG AGTTAATTTATGATTGATA AUUG TTTATTA IL6_827 AAUGUUGG 1403 AGAACTGACAATATGAAT 1603 mouse GACACUAUU GTTGGGACACTATTTTAAT UUA TATTTTTA IL6_1029 GUUUACCUC 1404 TCTTGGAATGTATAAGTTT 1604 mouse AAUGAAUU ACCTCAATGAATTGCTAA GCU TTTAAATA IL6_1016 UUGGAAUG 1405 TAGCCAGATGGTTTCTTGG 1605 mouse UAUAAGUU AATGTATAAGTTTACCTCA UACC ATGAATT IL6_779 UGUCAGGUA 1406 GAAAATATATCCTGTTGTC 1606 mouse UCUGACUUA AGGTATCTGACTTATGTTG UG TTCTCTA IL6_1066 AAAGAAAUC 1407 TTTAAATATGTTTTTAAAG 1607 mouse UUUGUGAU AAATCTTTGTGATGTATTT GUA TTATAAT IL6_854 UUAAUUUA 1408 CTATTTTAATTATTTTTAA 1608 mouse UUGAUAAU TTTATTGATAATTTAAATA UUAA AGTAAAC IL6_830 GUUGGGACA 1409 ACTGACAATATGAATGTT 1609 mouse CUAUUUUAA GGGACACTATTTTAATTAT UU TTTTAATT IL6_1089 UUAUAAUG 1410 CTTTGTGATGTATTTTTAT 1610 mouse UUUAGACUG AATGTTTAGACTGTCTTCA UCU AACAAAT IL6_931 UUGAAAUG 1411 TTTATGAAGTGTCACTTGA 1611 mouse UUAUAUGU AATGTTATATGTTATAGTT UAUA TTGAAAT IL6_538 AACCAAGAG 1412 CTAATTCATATCTTCAACC 1612 mouse GUAAAAGA AAGAGGTAAAAGATTTAC UUU ATAAAATA IL6_887 AAGUUAAU 1413 AAATAAGTAAACTTTAAG 1613 mouse UUAUGAUU TTAATTTATGATTGATATT GAUA TATTATTT IL6_408 AUUGAAAA 1414 TCAGGAAATTTGCCTATTG 1614 mouse UUUCCUCUG AAAATTTCCTCTGGTCTTC GUC TGGAGTA IL6_235 GGCUUAAUU 1415 ACTTCACAAGTCGGAGGC 1615 mouse ACACAUGUU TTAATTACACATGTTCTCT CU GGGAAATC IL6_725 CUAAGCAUA 1416 CCTAGTGCGTTATGCCTAA 1616 mouse UCAGUUUGU GCATATCAGTTTGTGGAC GG ATTCCTCA IL6_401 UUUGCCUAU 1417 GATATAATCAGGAAATTT 1617 mouse UGAAAAUU GCCTATTGAAAATTTCCTC UCC TGGTCTTC IL6_973 AUCUAUUUG 1418 AATGATAACCTAAAAATC 1618 mouse AUAUAAAU TATTTGATATAAATATTCT AUU GTTACCTA IL6_926 GUCACUUGA 1419 TTATTTTTATGAAGTGTCA 1619 mouse AAUGUUAU CTTGAAATGTTATATGTTA AUG TAGTTTT IL6_862 UUGAUAAU 1420 ATTATTTTTAATTTATTGA 1620 mouse UUAAAUAA TAATTTAAATAAGTAAAC GUAA TTTAAGTT IL6_868 AUUUAAAU 1421 TTTAATTTATTGATAATTT 1621 mouse AAGUAAACU AAATAAGTAAACTTTAAG UUA TTAATTTA IL6_869 UUUAAAUA 1422 TTAATTTATTGATAATTTA 1622 mouse AGUAAACUU AATAAGTAAACTTTAAGT UAA TAATTTAT IL6_885 UUAAGUUA 1423 TTAAATAAGTAAACTTTA 1623 mouse AUUUAUGA AGTTAATTTATGATTGATA UUGA TTTATTAT IL6_871 UAAAUAAG 1424 AATTTATTGATAATTTAAA 1624 mouse UAAACUUUA TAAGTAAACTTTAAGTTA AGU ATTTATGA IL6_652 UUCAUCUUG 1425 ACCAAGACCATCCAATTC 1625 mouse AAAUCACUU ATCTTGAAATCACTTGAA GA GAATTTCTA IL6_816 CUGACAAUA 1426 GTTCTCTACGAAGAACTG 1626 mouse UGAAUGUU ACAATATGAATGTTGGGA GGG CACTATTTT IL6_802 UUCUCUACG 1427 ATCTGACTTATGTTGTTCT 1627 mouse AAGAACUGA CTACGAAGAACTGACAAT CA ATGAATGT IL6_761 UCAGAAAAU 1428 CATTCCTCACTGTGGTCAG 1628 mouse AUAUCCUGU AAAATATATCCTGTTGTCA UG GGTATCT IL6_1007 GAUGGUUUC 1429 TCTGTTACCTAGCCAGATG 1629 mouse UUGGAAUG GTTTCTTGGAATGTATAAG UAU TTTACCT IL6_923 AGUGUCACU 1430 TTATTATTTTTATGAAGTG 1630 mouse UGAAAUGU TCACTTGAAATGTTATATG UAU TTATAGT IL6_978 UUUGAUAU 1431 TAACCTAAAAATCTATTTG 1631 mouse AAAUAUUCU ATATAAATATTCTGTTACC GUU TAGCCAG IL6_1063 UUUAAAGA 1432 TAATTTAAATATGTTTTTA 1632 mouse AAUCUUUGU AAGAAATCTTTGTGATGT GAU ATTTTTAT IL6_953 UUUGAAAU 1433 GTTATATGTTATAGTTTTG 1633 mouse GAUAACCUA AAATGATAACCTAAAAAT AAA CTATTTGA IL6_389 AUAAUCAGG 1434 GCTACCAAACTGGATATA 1634 mouse AAAUUUGCC ATCAGGAAATTTGCCTATT UA GAAAATTT IL6_541 CAAGAGGUA 1435 ATTCATATCTTCAACCAAG 1635 mouse AAAGAUUU AGGTAAAAGATTTACATA ACA AAATAGTC IL6_268 GAAAUGAG 1436 CTCTGGGAAATCGTGGAA 1636 mouse AAAAGAGU ATGAGAAAAGAGTTGTGC UGUG AATGGCAAT IL6_880 AAACUUUAA 1437 ATAATTTAAATAAGTAAA 1637 mouse GUUAAUUU CTTTAAGTTAATTTATGAT AUG TGATATTT IL6_861 AUUGAUAA 1438 AATTATTTTTAATTTATTG 1638 mouse UUUAAAUA ATAATTTAAATAAGTAAA AGUA CTTTAAGT IL6_875 UAAGUAAAC 1439 TATTGATAATTTAAATAA 1639 mouse UUUAAGUU GTAAACTTTAAGTTAATTT AAU ATGATTGA IL6_256 UGGGAAAUC 1440 ATTACACATGTTCTCTGGG 1640 mouse GUGGAAAU AAATCGTGGAAATGAGAA GAG AAGAGTTG IL6_351 AGAGAUACA 1441 CAATCTGAAACTTCCAGA 1641 mouse AAGAAAUG GATACAAAGAAATGATGG AUG ATGCTACCA IL6_824 AUGAAUGU 1442 CGAAGAACTGACAATATG 1642 mouse UGGGACACU AATGTTGGGACACTATTTT AUU AATTATTT IL6_332 AAAACAAUC 1443 ATGATGCACTTGCAGAAA 1643 mouse UGAAACUUC ACAATCTGAAACTTCCAG CA AGATACAAA IL6_403 UGCCUAUUG 1444 TATAATCAGGAAATTTGC 1644 mouse AAAAUUUCC CTATTGAAAATTTCCTCTG UC GTCTTCTG IL6_762 CAGAAAAUA 1445 ATTCCTCACTGTGGTCAGA 1645 mouse UAUCCUGUU AAATATATCCTGTTGTCAG GU GTATCTG IL6_879 UAAACUUUA 1446 GATAATTTAAATAAGTAA 1646 mouse AGUUAAUU ACTTTAAGTTAATTTATGA UAU TTGATATT IL6_947 UAUAGUUU 1447 TGAAATGTTATATGTTATA 1647 mouse UGAAAUGA GTTTTGAAATGATAACCT UAAC AAAAATCT IL6_649 CAAUUCAUC 1448 AGGACCAAGACCATCCAA 1648 mouse UUGAAAUCA TTCATCTTGAAATCACTTG CU AAGAATTT IL6_897 AUGAUUGA 1449 ACTTTAAGTTAATTTATGA 1649 mouse UAUUUAUU TTGATATTTATTATTTTTA AUUU TGAAGTG IL6_1043 AUUGCUAAU 1450 AGTTTACCTCAATGAATTG 1650 mouse UUAAAUAU CTAATTTAAATATGTTTTT GUU AAAGAAA IL6_576 UACCCCAAU 1451 TAAAATAGTCCTTCCTACC 1651 mouse UUCCAAUGC CCAATTTCCAATGCTCTCC UC TAACAGA IL6_90 CUCUGCAAG 1452 CGCTATGAAGTTCCTCTCT 1652 mouse AGACUUCCA GCAAGAGACTTCCATCCA UC GTTGCCTT IL6_378 CCAAACUGG 1453 AAATGATGGATGCTACCA 1653 mouse AUAUAAUCA AACTGGATATAATCAGGA GG AATTTGCCT IL6_853 UUUAAUUU 1454 ACTATTTTAATTATTTTTA 1654 mouse AUUGAUAA ATTTATTGATAATTTAAAT UUUA AAGTAAA IL6_367 GAUGGAUGC 1455 GAGATACAAAGAAATGAT 1655 mouse UACCAAACU GGATGCTACCAAACTGGA GG TATAATCAG IL6_992 UCUGUUACC 1456 ATTTGATATAAATATTCTG 1656 mouse UAGCCAGAU TTACCTAGCCAGATGGTTT GG CTTGGAA IL6_939 UUAUAUGU 1457 GTGTCACTTGAAATGTTAT 1657 mouse UAUAGUUU ATGTTATAGTTTTGAAATG UGAA ATAACCT IL6_1037 CAAUGAAUU 1458 TGTATAAGTTTACCTCAAT 1658 mouse GCUAAUUUA GAATTGCTAATTTAAATAT AA GTTTTTA IL6_661 AAAUCACUU 1459 ATCCAATTCATCTTGAAAT 1659 mouse GAAGAAUU CACTTGAAGAATTTCTAA UCU AAGTCACT IL6_1042 AAUUGCUAA 1460 AAGTTTACCTCAATGAATT 1660 mouse UUUAAAUA GCTAATTTAAATATGTTTT UGU TAAAGAA IL6_995 GUUACCUAG 1461 TGATATAAATATTCTGTTA 1661 mouse CCAGAUGGU CCTAGCCAGATGGTTTCTT UU GGAATGT IL6_1017 UGGAAUGU 1462 AGCCAGATGGTTTCTTGG 1662 mouse AUAAGUUU AATGTATAAGTTTACCTCA ACCU ATGAATTG IL6_929 ACUUGAAAU 1463 TTTTTATGAAGTGTCACTT 1663 mouse GUUAUAUG GAAATGTTATATGTTATA UUA GTTTTGAA IL6_878 GUAAACUUU 1464 TGATAATTTAAATAAGTA 1664 mouse AAGUUAAU AACTTTAAGTTAATTTATG UUA ATTGATAT IL6_945 GUUAUAGU 1465 CTTGAAATGTTATATGTTA 1665 mouse UUUGAAAU TAGTTTTGAAATGATAAC GAUA CTAAAAAT IL6_285 GUGCAAUGG 1466 AATGAGAAAAGAGTTGTG 1666 mouse CAAUUCUGA CAATGGCAATTCTGATTGT UU ATGAACAA IL6_662 AAUCACUUG 1467 TCCAATTCATCTTGAAATC 1667 mouse AAGAAUUUC ACTTGAAGAATTTCTAAA UA AGTCACTT IL6_852 UUUUAAUU 1468 CACTATTTTAATTATTTTT 1668 mouse UAUUGAUA AATTTATTGATAATTTAAA AUUU TAAGTAA IL6_348 UCCAGAGAU 1469 AAACAATCTGAAACTTCC 1669 mouse ACAAAGAAA AGAGATACAAAGAAATGA UG TGGATGCTA IL6_883 CUUUAAGUU 1470 ATTTAAATAAGTAAACTTT 1670 mouse AAUUUAUG AAGTTAATTTATGATTGAT AUU ATTTATT IL6_1033 ACCUCAAUG 1471 GGAATGTATAAGTTTACC 1671 mouse AAUUGCUAA TCAATGAATTGCTAATTTA UU AATATGTT IL6_758 UGGUCAGAA 1472 GGACATTCCTCACTGTGGT 1672 mouse AAUAUAUCC CAGAAAATATATCCTGTT UG GTCAGGTA IL6_1090 UAUAAUGU 1473 TTTGTGATGTATTTTTATA 1673 mouse UUAGACUGU ATGTTTAGACTGTCTTCAA CUU ACAAATA IL6_979 UUGAUAUA 1474 AACCTAAAAATCTATTTG 1674 mouse AAUAUUCUG ATATAAATATTCTGTTACC UUA TAGCCAGA IL6_916 UUUAUGAA 1475 TTGATATTTATTATTTTTA 1675 mouse GUGUCACUU TGAAGTGTCACTTGAAAT GAA GTTATATG IL6_328 GCAGAAAAC 1476 AACGATGATGCACTTGCA 1676 mouse AAUCUGAAA GAAAACAATCTGAAACTT CU CCAGAGATA IL6_831 UUGGGACAC 1477 CTGACAATATGAATGTTG 1677 mouse UAUUUUAA GGACACTATTTTAATTATT UUA TTTAATTT IL6_385 GGAUAUAA 1478 GGATGCTACCAAACTGGA 1678 mouse UCAGGAAAU TATAATCAGGAAATTTGC UUG CTATTGAAA IL6_1087 UUUUAUAA 1479 ATCTTTGTGATGTATTTTT 1679 mouse UGUUUAGAC ATAATGTTTAGACTGTCTT UGU CAAACAA IL6_249 UGUUCUCUG 1480 AGGCTTAATTACACATGTT 1680 mouse GGAAAUCGU CTCTGGGAAATCGTGGAA GG ATGAGAAA IL6_670 GAAGAAUU 1481 ATCTTGAAATCACTTGAA 1681 mouse UCUAAAAGU GAATTTCTAAAAGTCACTT CAC TGAGATCT IL6_653 UCAUCUUGA 1482 CCAAGACCATCCAATTCA 1682 mouse AAUCACUUG TCTTGAAATCACTTGAAG AA AATTTCTAA IL6_283 UUGUGCAAU 1483 GAAATGAGAAAAGAGTTG 1683 mouse GGCAAUUCU TGCAATGGCAATTCTGATT GA GTATGAAC IL6_532 AUCUUCAAC 1484 GAAACTCTAATTCATATCT 1684 mouse CAAGAGGUA TCAACCAAGAGGTAAAAG AA ATTTACAT IL6_996 UUACCUAGC 1485 GATATAAATATTCTGTTAC 1685 mouse CAGAUGGUU CTAGCCAGATGGTTTCTTG UC GAATGTA IL6_1032 UACCUCAAU 1486 TGGAATGTATAAGTTTAC 1686 mouse GAAUUGCUA CTCAATGAATTGCTAATTT AU AAATATGT IL6_657 CUUGAAAUC 1487 GACCATCCAATTCATCTTG 1687 mouse ACUUGAAGA AAATCACTTGAAGAATTT AU CTAAAAGT IL6_895 UUAUGAUU 1488 AAACTTTAAGTTAATTTAT 1688 mouse GAUAUUUA GATTGATATTTATTATTTT UUAU TATGAAG IL6_400 AUUUGCCUA 1489 GGATATAATCAGGAAATT 1689 mouse UUGAAAAU TGCCTATTGAAAATTTCCT UUC CTGGTCTT IL6_1014 UCUUGGAAU 1490 CCTAGCCAGATGGTTTCTT 1690 mouse GUAUAAGU GGAATGTATAAGTTTACC UUA TCAATGAA IL6_866 UAAUUUAA 1491 TTTTTAATTTATTGATAAT 1691 mouse AUAAGUAA TTAAATAAGTAAACTTTA ACUU AGTTAATT IL6_339 UCUGAAACU 1492 ACTTGCAGAAAACAATCT 1692 mouse UCCAGAGAU GAAACTTCCAGAGATACA AC AAGAAATGA IL6_233 GAGGCUUAA 1493 CCACTTCACAAGTCGGAG 1693 mouse UUACACAUG GCTTAATTACACATGTTCT UU CTGGGAAA IL6_877 AGUAAACUU 1494 TTGATAATTTAAATAAGT 1694 mouse UAAGUUAA AAACTTTAAGTTAATTTAT UUU GATTGATA

TABLE 11C Host target FURIN - 20 nucleotide targets and 45 nucleotide gene target regions SEQ SEQ SEQUENCE ID ID ID 20 nt Sequence NO: 45 nt Gene Region NO: Species FURIN_4183 AUGGACAUGAG 1695 TGCTGGTTCTATTTAATGGACAT 1795 human AUAAUGUUA GAGATAATGTTAGAGGTTTTAA FURIN_3561 UUUAGAUGCUG 1696 TTGTGATTATTTCACTTTAGATG 1796 human AUGAUUUGU CTGATGATTTGTTTTTGTATTT FURIN_3555 UUUCACUUUAG 1697 TTCACTTTGTGATTATTTCACTTT 1797 human AUGCUGAUG AGATGCTGATGATTTGTTTTT FURIN_3563 UAGAUGCUGAU 1698 GTGATTATTTCACTTTAGATGCT 1798 human GAUUUGUUU GATGATTTGTTTTTGTATTTTT FURIN_3529 GAGGAUAUAUU 1699 TCTCAGGGGCTGTTTGAGGATAT 1799 human UUCACUUUG ATTTTCACTTTGTGATTATTTC FURIN_4161 CCAGCAUUGCU 1700 GTAATTTAAACAGGCCCAGCATT 1800 human GGUUCUAUU GCTGGTTCTATTTAATGGACAT FURIN_3537 AUUUUCACUUU 1701 GCTGTTTGAGGATATATTTTCAC 1801 human GUGAUUAUU TTTGTGATTATTTCACTTTAGA FURIN_1873 GCCUUCAUGAC 1702 GGGTTTAATGACTGGGCCTTCAT 1802 human AACUCAUUC GACAACTCATTCCTGGGATGAG FURIN_3545 UUUGUGAUUAU 1703 AGGATATATTTTCACTTTGTGAT 1803 human UUCACUUUA TATTTCACTTTAGATGCTGATG FURIN_3525 GUUUGAGGAUA 1704 GGGATCTCAGGGGCTGTTTGAGG 1804 human UAUUUUCAC ATATATTTTCACTTTGTGATTA FURIN_4184 UGGACAUGAGA 1705 GCTGGTTCTATTTAATGGACATG 1805 human UAAUGUUAG AGATAATGTTAGAGGTTTTAAA FURIN_3995 UCUGGGAGUCC 1706 ATCAGTCCCCTCCCATCTGGGAG 1806 human CCUUUUCUU TCCCCTTTTCTTTTCTACCCTA FURIN_4152 UAAACAGGCCC 1707 TTTTTTCTTGTAATTTAAACAGG 1807 human AGCAUUGCU CCCAGCATTGCTGGTTCTATTT FURIN_1878 CAUGACAACUC 1708 TAATGACTGGGCCTTCATGACAA 1808 human AUUCCUGGG CTCATTCCTGGGATGAGGATCC FURIN_4192 AGAUAAUGUUA 1709 TATTTAATGGACATGAGATAATG 1809 human GAGGUUUUA TTAGAGGTTTTAAAGTGATTAA FURIN_1969 ACCAAGUUCAC 1710 AACTATGGGACGCTGACCAAGTT 1810 human CCUCGUACU CACCCTCGTACTCTATGGCACC FURIN_312 AGCAGCAACAG 1711 GTTGCTATGGGTGGTAGCAGCAA 1811 human GAACCUUGG CAGGAACCTTGGTCCTGCTAGC FURIN_3538 UUUUCACUUUG 1712 CTGTTTGAGGATATATTTTCACT 1812 human UGAUUAUUU TTGTGATTATTTCACTTTAGAT FURIN_4193 GAUAAUGUUAG 1713 ATTTAATGGACATGAGATAATGT 1813 human AGGUUUUAA TAGAGGTTTTAAAGTGATTAAA FURIN_3957 UUUGCACCCCU 1714 TCTTCTGACGTGCCTTTTGCACC 1814 human CCCAUUAGG CCTCCCATTAGGACAATCAGTC FURIN_1925 UAGAGAUUGAA 1715 CTGGCGAGTGGGTCCTAGAGATT 1815 human AACACCAGC GAAAACACCAGCGAAGCCAACA FURIN_2894 AAAGGAGUGAA 1716 CCCTTCCATGTGGAGAAAGGAGT 1816 human ACCUUUAGG GAAACCTTTAGGGCAGCTTGCC FURIN_4117 CUGGGUUGGUG 1717 TGGTTTTGTAAGATGCTGGGTTG 1817 human CACAGUGAU GTGCACAGTGATTTTTTTCTTG FURIN_3534 UAUAUUUUCAC 1718 GGGGCTGTTTGAGGATATATTTT 1818 human UUUGUGAUU CACTTTGTGATTATTTCACTTT FURIN_4150 UUUAAACAGGC 1719 ATTTTTTTCTTGTAATTTAAACAG 1819 human CCAGCAUUG GCCCAGCATTGCTGGTTCTAT FURIN_3929 CCCUCAAACCU 1720 TGACCTGTCATGCCCCCCTCAAA 1820 human CCUCUUCUG CCTCCTCTTCTGACGTGCCTTT FURIN_3560 CUUUAGAUGCU 1721 TTTGTGATTATTTCACTTTAGATG 1821 human GAUGAUUUG CTGATGATTTGTTTTTGTATT FURIN_4100 GCUGGUUUUGU 1722 CTCGTGGCCAGCCCGGCTGGTTT 1822 human AAGAUGCUG TGTAAGATGCTGGGTTGGTGCA FURIN_712 CACGGCAUUGU 1723 CAGGGCTACACAGGGCACGGCA 1823 human GGUCUCCAU TTGTGGTCTCCATTCTGGACGAT FURIN_3541 UCACUUUGUGA 1724 TTTGAGGATATATTTTCACTTTGT 1824 human UUAUUUCAC GATTATTTCACTTTAGATGCT FURIN_4169 GCUGGUUCUAU 1725 AACAGGCCCAGCATTGCTGGTTC 1825 human UUAAUGGAC TATTTAATGGACATGAGATAAT FURIN_1303 AGCAGUGGCAA 1726 CTGGCCACGACCTACAGCAGTG 1826 human CCAGAAUGA GCAACCAGAATGAGAAGCAGATC FURIN_765 CUUGGCAGGCA 1727 GAAGAACCACCCGGACTTGGCA 1827 human AUUAUGAUC GGCAATTATGATCCTGGGGCCAG FURIN_1942 AGCGAAGCCAA 1728 GAGATTGAAAACACCAGCGAAG 1828 human CAACUAUGG CCAACAACTATGGGACGCTGACC FURIN_1945 GAAGCCAACAA 1729 ATTGAAAACACCAGCGAAGCCA 1829 human CUAUGGGAC ACAACTATGGGACGCTGACCAAG FURIN_2780 GGAGACUGCUU 1730 GGAGGCAAGAGGGGTGGAGACT 1830 human CCCAUCCUA GCTTCCCATCCTACCCTCGGGCC FURIN_3827 UCAUAGGUCAC 1731 CGCCATGCCGGGGGTTCATAGGT 1831 human UGGCUCUCC CACTGGCTCTCCAAGTGCCAGA FURIN_3547 UGUGAUUAUUU 1732 GATATATTTTCACTTTGTGATTAT 1832 human CACUUUAGA TTCACTTTAGATGCTGATGAT FURIN_3559 ACUUUAGAUGC 1733 CTTTGTGATTATTTCACTTTAGAT 1833 human UGAUGAUUU GCTGATGATTTGTTTTTGTAT FURIN_4177 UAUUUAAUGGA 1734 CAGCATTGCTGGTTCTATTTAAT 1834 human CAUGAGAUA GGACATGAGATAATGTTAGAGG FURIN_2158 UAUAGCACCGA 1735 GTCCTCGATACGCACTATAGCAC 1835 human GAAUGACGU CGAGAATGACGTGGAGACCATC FURIN_4165 CAUUGCUGGUU 1736 TTTAAACAGGCCCAGCATTGCTG 1836 human CUAUUUAAU GTTCTATTTAATGGACATGAGA FURIN_4186 GACAUGAGAUA 1737 TGGTTCTATTTAATGGACATGAG 1837 human AUGUUAGAG ATAATGTTAGAGGTTTTAAAGT FURIN_779 AUGAUCCUGGG 1738 ACTTGGCAGGCAATTATGATCCT 1838 human GCCAGUUUU GGGGCCAGTTTTGATGTCAATG FURIN_1871 GGGCCUUCAUG 1739 ATGGGTTTAATGACTGGGCCTTC 1839 human ACAACUCAU ATGACAACTCATTCCTGGGATG FURIN_3552 UUAUUUCACUU 1740 ATTTTCACTTTGTGATTATTTCAC 1840 human UAGAUGCUG TTTAGATGCTGATGATTTGTT FURIN_4180 UUAAUGGACAU 1741 CATTGCTGGTTCTATTTAATGGA 1841 human GAGAUAAUG CATGAGATAATGTTAGAGGTTT FURIN_1429 GCCAAUAAGAA 1742 GCTCTCACCCTGGAGGCCAATAA 1842 human CCUCACAUG GAACCTCACATGGCGGGACATG FURIN_1654 AAAGACAUCGG 1743 ATCCTCACCGAGCCCAAAGACAT 1843 human GAAACGGCU CGGGAAACGGCTCGAGGTGCGG FURIN_3968 CCCAUUAGGAC 1744 GCCTTTTGCACCCCTCCCATTAG 1844 human AAUCAGUCC GACAATCAGTCCCCTCCCATCT FURIN_1855 GAUGGGUUUAA 1745 CATGACTACTCCGCAGATGGGTT 1845 human UGACUGGGC TAATGACTGGGCCTTCATGACA FURIN_3513 AUCUCAGGGGC 1746 CTTTCCCCTGTGGGGATCTCAGG 1846 human UGUUUGAGG GGCTGTTTGAGGATATATTTTC FURIN_4108 UGUAAGAUGCU 1747 CAGCCCGGCTGGTTTTGTAAGAT 1847 human GGGUUGGUG GCTGGGTTGGTGCACAGTGATT FURIN_4170 CUGGUUCUAUU 1748 ACAGGCCCAGCATTGCTGGTTCT 1848 human UAAUGGACA ATTTAATGGACATGAGATAATG FURIN_367 UUCACCAACAC 1749 CAGGGCCAGAAGGTCTTCACCA 1849 human GUGGGCUGU ACACGTGGGCTGTGCGCATCCCT FURIN_2492 UGCGCUCUGGC 1750 TCCTGGTCCTGCAGCTGCGCTCT 1850 human UUUAGUUUU GGCTTTAGTTTTCGGGGGGTGA FURIN_2882 UUCCAUGUGGA 1751 CACCCTCAGCACCCCTTCCATGT 1851 human GAAAGGAGU GGAGAAAGGAGTGAAACCTTTA FURIN_4175 UCUAUUUAAUG 1752 CCCAGCATTGCTGGTTCTATTTA 1852 human GACAUGAGA ATGGACATGAGATAATGTTAGA FURIN_4201 UAGAGGUUUUA 1753 GACATGAGATAATGTTAGAGGTT 1853 human AAGUGAUUA TTAAAGTGATTAAACGTGCAGA FURIN_749 AGAAGAACCAC 1754 TGGACGATGGCATCGAGAAGAA 1854 human CCGGACUUG CCACCCGGACTTGGCAGGCAATT FURIN_3523 CUGUUUGAGGA 1755 TGGGGATCTCAGGGGCTGTTTGA 1855 human UAUAUUUUC GGATATATTTTCACTTTGTGAT FURIN_1856 AUGGGUUUAAU 1756 ATGACTACTCCGCAGATGGGTTT 1856 human GACUGGGCC AATGACTGGGCCTTCATGACAA FURIN_1859 GGUUUAAUGAC 1757 ACTACTCCGCAGATGGGTTTAAT 1857 human UGGGCCUUC GACTGGGCCTTCATGACAACTC FURIN_2638 UUUAUCAAAGA 1758 GGCGAGAGGACCGCCTTTATCA 1858 human CCAGAGCGC AAGACCAGAGCGCCCTCTGATGA FURIN_3516 UCAGGGGCUGU 1759 TCCCCTGTGGGGATCTCAGGGGC 1859 human UUGAGGAUA TGTTTGAGGATATATTTTCACT FURIN_3554 AUUUCACUUUA 1760 TTTCACTTTGTGATTATTTCACTT 1860 human GAUGCUGAU TAGATGCTGATGATTTGTTTT FURIN_1936 AACACCAGCGA 1761 GTCCTAGAGATTGAAAACACCA 1861 human AGCCAACAA GCGAAGCCAACAACTATGGGACG FURIN_2458 CUGGUCUUCGU 1762 TGCGCCTTCATCGTGCTGGTCTT 1862 human CACUGUCUU CGTCACTGTCTTCCTGGTCCTG FURIN_313 GCAGCAACAGG 1763 TTGCTATGGGTGGTAGCAGCAAC 1863 human AACCUUGGU AGGAACCTTGGTCCTGCTAGCA FURIN_2520 GAAGGUGUACA 1764 TAGTTTTCGGGGGGTGAAGGTGT 1864 human CCAUGGACC ACACCATGGACCGTGGCCTCAT FURIN_1310 GCAACCAGAAU 1765 CGACCTACAGCAGTGGCAACCA 1865 human GAGAAGCAG GAATGAGAAGCAGATCGTGACGA FURIN_1752 CACCCUGUCCU 1766 CGCTCAGGCGCGGCTCACCCTGT 1866 human AUAAUCGCC CCTATAATCGCCGTGGCGACCT FURIN_4166 AUUGCUGGUUC 1767 TTAAACAGGCCCAGCATTGCTGG 1867 human UAUUUAAUG TTCTATTTAATGGACATGAGAT FURIN_834 GUACACACAGA 1768 TGACCCCCAGCCTCGGTACACAC 1868 human UGAAUGACA AGATGAATGACAACAGGCACGG FURIN_3517 CAGGGGCUGUU 1769 CCCCTGTGGGGATCTCAGGGGCT 1869 human UGAGGAUAU GTTTGAGGATATATTTTCACTT FURIN_3550 GAUUAUUUCAC 1770 ATATTTTCACTTTGTGATTATTTC 1870 human UUUAGAUGC ACTTTAGATGCTGATGATTTG FURIN_2694 UCCCCUCCUUG 1771 ACCCCCTCAAGCCAATCCCCTCC 1871 human GGCACUUUU TTGGGCACTTTTTAATTCACCA FURIN_578 UGGCAAAGCGA 1772 GGCTGGAACAGCAGGTGGCAAA 1872 human CGGACUAAA GCGACGGACTAAACGGGACGTGT FURIN_1246 UUUGGCAACGU 1773 AGCAGCGCCACGCAGTTTGGCA 1873 human GCCGUGGUA ACGTGCCGTGGTACAGCGAGGCC FURIN_1425 GGAGGCCAAUA 1774 CATTGCTCTCACCCTGGAGGCCA 1874 human AGAACCUCA ATAAGAACCTCACATGGCGGGA FURIN_1018 AUCCACAUCUA 1775 CTGAACCCCAACCACATCCACAT 1875 human CAGUGCCAG CTACAGTGCCAGCTGGGGCCCC FURIN_1858 GGGUUUAAUGA 1776 GACTACTCCGCAGATGGGTTTAA 1876 human CUGGGCCUU TGACTGGGCCTTCATGACAACT FURIN_1924 CUAGAGAUUGA 1777 TCTGGCGAGTGGGTCCTAGAGAT 1877 human AAACACCAG TGAAAACACCAGCGAAGCCAAC FURIN_293 CCUGGUUGCUA 1778 CCATGGAGCTGAGGCCCTGGTTG 1878 human UGGGUGGUA CTATGGGTGGTAGCAGCAACAG FURIN_790 GCCAGUUUUGA 1779 AATTATGATCCTGGGGCCAGTTT 1879 human UGUCAAUGA TGATGTCAATGACCAGGACCCT FURIN_962 AUGGCGAGGUG 1780 GGGTGCGCATGCTGGATGGCGA 1880 human ACAGAUGCA GGTGACAGATGCAGTGGAGGCAC FURIN_1757 UGUCCUAUAAU 1781 AGGCGCGGCTCACCCTGTCCTAT 1881 human CGCCGUGGC AATCGCCGTGGCGACCTGGCCA FURIN_1848 CUCCGCAGAUG 1782 CAGGCCACATGACTACTCCGCAG 1882 human GGUUUAAUG ATGGGTTTAATGACTGGGCCTT FURIN_3020 GUCCCUCUAAA 1783 CTCTTGCCCTTCCCTGTCCCTCTA 1883 human GCAAUAAUG AAGCAATAATGGTCCCATCCA FURIN_3511 GGAUCUCAGGG 1784 TGCTTTCCCCTGTGGGGATCTCA 1884 human GCUGUUUGA GGGGCTGTTTGAGGATATATTT FURIN_760 CCGGACUUGGC 1785 ATCGAGAAGAACCACCCGGACT 1885 human AGGCAAUUA TGGCAGGCAATTATGATCCTGGG FURIN_770 CAGGCAAUUAU 1786 ACCACCCGGACTTGGCAGGCAA 1886 human GAUCCUGGG TTATGATCCTGGGGCCAGTTTTG FURIN_827 AGCCUCGGUAC 1787 AGGACCCTGACCCCCAGCCTCGG 1887 human ACACAGAUG TACACACAGATGAATGACAACA FURIN_1199 GCUACACCAAC 1788 GCTGCAACTGCGACGGCTACACC 1888 human AGUAUCUAC AACAGTATCTACACGCTGTCCA FURIN_2459 UGGUCUUCGUC 1789 GCGCCTTCATCGTGCTGGTCTTC 1889 human ACUGUCUUC GTCACTGTCTTCCTGGTCCTGC FURIN_4106 UUUGUAAGAUG 1790 GCCAGCCCGGCTGGTTTTGTAAG 1890 human CUGGGUUGG ATGCTGGGTTGGTGCACAGTGA FURIN_4173 GUUCUAUUUAA 1791 GGCCCAGCATTGCTGGTTCTATT 1891 human UGGACAUGA TAATGGACATGAGATAATGTTA FURIN_4199 GUUAGAGGUUU 1792 TGGACATGAGATAATGTTAGAG 1892 human UAAAGUGAU GTTTTAAAGTGATTAAACGTGCA FURIN_773 GCAAUUAUGAU 1793 ACCCGGACTTGGCAGGCAATTAT 1893 human CCUGGGGCC GATCCTGGGGCCAGTTTTGATG FURIN_786 UGGGGCCAGUU 1794 AGGCAATTATGATCCTGGGGCCA 1894 human UUGAUGUCA GTTTTGATGTCAATGACCAGGA

TABLE 11D Host target ACE2-20 nucleotide targets and 45 nucleotide gene target regions SE- SEQ SEQ QUENCE ID ID ID 20 nt Sequence NO: 45 nt Gene Region NO: Species ACE2_ UUGGAUUUCAUAC 1895 TGACATAGATACTCTTTGGATTT 2095 human 52 CAUGUGG CATACCATGTGGAGGCTTTCTT ACE2_ UGUCAAAACUAUG 1896 AAAGATATCATTAAATGTCAAA 2096 human 2730 ACUCUGU ACTATGACTCTGTTCAGAAAAAA ACE2_ CCUAGCAUUGGAA 1897 ATCAGAACCCTGGACCCTAGCAT 2097 human 1918 AAUGUUG TGGAAAATGTTGTAGGAGCAAA ACE2_ AUGUAAAUGUUAA 1898 AGGTGATTTTGTTGTATGTAAAT 2098 human 2674 UUUCAUG GTTAATTTCATGGTATAGAAAA ACE2_ UCUGUUUCUUAAU 1899 GGATTTGACTTCTGTTCTGTTTCT 2099 human 2849 AAGGAUU TAATAAGGATTTTGTATTAGA ACE2_ CAGGAGUUGACAU 1900 ATGGCTACAGAGGATCAGGAGT 2100 human 30 AGAUACU TGACATAGATACTCTTTGGATTT ACE2_ AAUGAUUACUCAU 1901 CTGTTCCATGTTTCTAATGATTA 2101 human 1739 UCAUUCG CTCATTCATTCGATATTACACA ACE2_ AUCGAUAUUAGCA 1902 AATCCTTATGCCTCCATCGATAT 2102 human 2567 AAGGAGA TAGCAAAGGAGAAAATAATCCA ACE2_ CAUGGUAUAGAAA 1903 TGTAAATGTTAATTTCATGGTAT 2103 human 2690 AUAUAAG AGAAAATATAAGATGATAAAGA ACE2_ UUUGAAACCAAGA 1904 TGTGCGAGTGGCTAATTTGAAAC 2104 human 2239 AUCUCCU CAAGAATCTCCTTTAATTTCTT ACE2_ AUGGGAGUGAUAG 1905 GTTTTTGGAGTTGTGATGGGAGT 2105 human 2465 UGGUUGG GATAGTGGTTGGCATTGTCATC ACE2_ UUGAAGAGAUUAA 1906 ATGTGGAACATACCTTTGAAGAG 2106 human 906 ACCAUUA ATTAAACCATTATATGAACATC ACE2_ UUUGUUGUAUGUA 1907 TCCTCTTGAGGTGATTTTGTTGT 2107 human 2666 AAUGUUA ATGTAAATGTTAATTTCATGGT ACE2_ UUCAUGGUAUAGA 1908 TATGTAAATGTTAATTTCATGGT 2108 human 2688 AAAUAUA ATAGAAAATATAAGATGATAAA ACE2_ CUCUGGAUUUGAC 1909 AGTATTTATTTCTGTCTCTGGATT 2109 human 2830 UUCUGUU TGACTTCTGTTCTGTTTCTTA ACE2_ UAAACCAUUAUAU 1910 TACCTTTGAAGAGATTAAACCAT 2110 human 916 GAACAUC TATATGAACATCTTCATGCCTA ACE2_ UUCCUCUUGAGGU 1911 GAAAAATCTATGTTTTTCCTCTT 2111 human 2650 GAUUUUG GAGGTGATTTTGTTGTATGTAA ACE2_ CAUUAAAUGUCAA 1912 AGATGATAAAGATATCATTAAAT 2112 human 2723 AACUAUG GTCAAAACTATGACTCTGTTCA ACE2_ GGAGAAAAUAAUC 1913 ATCGATATTAGCAAAGGAGAAA 2113 human 2582 CAGGAUU ATAATCCAGGATTCCAAAACACT ACE2_ UGGCCAAGGAGAG 1914 TGTCCAAAGACAACATGGCCAA 2114 human 2778 AGCAUCU GGAGAGAGCATCTTCATTGACAT ACE2_ UCAGGGAUAAUCU 1915 GGTCTCACAGGCTGTTCAGGGAT 2115 human 2918 AAAUGUA AATCTAAATGTAAATGTCTGTT ACE2_ UUCAAGAAGACAA 1916 TTCTGTCACCCGATTTTCAAGAA 2116 human 1500 UGAAACA GACAATGAAACAGAAATAAACT ACE2_ AGGACCCUUUACC 1917 ATTCGATATTACACAAGGACCCT 2117 human 1769 AAUUCCA TTACCAATTCCAGTTTCAAGAA ACE2_ UGUAGGAGCAAAG 1918 AGCATTGGAAAATGTTGTAGGA 2118 human 1936 AACAUGA GCAAAGAACATGAATGTAAGGCC ACE2_ AGGAGCAAAGAAC 1919 ATTGGAAAATGTTGTAGGAGCA 2119 human 1939 AUGAAUG AAGAACATGAATGTAAGGCCACT ACE2_ UGGAGAAAAUCCU 1920 AAATAAAGCAAGAAGTGGAGAA 2120 human 2545 UAUGCCU AATCCTTATGCCTCCATCGATAT ACE2_ GGUGAUUUUGUUG 1921 TGTTTTTCCTCTTGAGGTGATTTT 2121 human 2660 UAUGUAA GTTGTATGTAAATGTTAATTT ACE2_ UUCUGCAGCCACA 1922 GGAAATCATGTCACTTTCTGCAG 2122 human 1447 CCUAAGC CCACACCTAAGCATTTAAAATC ACE2_ AAAACUAUGACUC 1923 ATATCATTAAATGTCAAAACTAT 2123 human 2734 UGUUCAG GACTCTGTTCAGAAAAAAAATT ACE2_ AUCUAAAUGUAAA 1924 GGCTGTTCAGGGATAATCTAAAT 2124 human 2927 UGUCUGU GTAAATGTCTGTTGAATTTCTG ACE2_ UUAGUCUAGGGAA 1925 TCTCATGAGGAGGTTTTAGTCTA 2125 human 167 AGUCAUU GGGAAAGTCATTCAGTGGATGT ACE2_ GGAGCAAGUGUUG 1926 CAAGGATATATCATTGGAGCAA 2126 human 2982 GAUCUUG GTGTTGGATCTTGTATGGAATAT ACE2_ UUUGACUUCUGUU 1927 ATTTCTGTCTCTGGATTTGACTTC 2127 human 2837 CUGUUUC TGTTCTGTTTCTTAATAAGGA ACE2_ UUUUGUAUUAGAG 1928 GTTTCTTAATAAGGATTTTGTAT 2128 human 2867 UAUAUUA TAGAGTATATTAGGGAAAGTGT ACE2_ GAGGCCAUUAUAU 1929 GGTCGGCAAGCAGCTGAGGCCA 2129 human 745 GAAGAGU TTATATGAAGAGTATGTGGTCTT ACE2_ CCAAGAAUCUCCU 1930 GTGGCTAATTTGAAACCAAGAAT 2130 human 2246 UUAAUUU CTCCTTTAATTTCTTTGTCACT ACE2_ GAUGAUGUUCAGA 1931 GGATTCCAAAACACTGATGATGT 2131 human 2612 CCUCCUU TCAGACCTCCTTTTAGAAAAAT ACE2_ UCUGGAUUUGACU 1932 GTATTTATTTCTGTCTCTGGATTT 2132 human 2831 UCUGUUC GACTTCTGTTCTGTTTCTTAA ACE2_ UCCACCAUUGAGG 1933 GTAACTGCTGCTCAGTCCACCAT 2133 human 272 AACAGGC TGAGGAACAGGCCAAGACATTT ACE2_ UUAUUACUUGAAC 1934 AATCCACAAGAATGCTTATTACT 2134 human 641 CAGGUUU TGAACCAGGTTTGAATGAAATA ACE2_ UGAACCAGGUUUG 1935 AGAATGCTTATTACTTGAACCAG 2135 human 649 AAUGAAA GTTTGAATGAAATAATGGCAAA ACE2_ UGUGGGAUGGAGU 1936 GAACAAGAATTCTTTTGTGGGAT 2136 human 2026 ACCGACU GGAGTACCGACTGGAGTCCATA ACE2_ UGAGGUGAUUUUG 1937 CTATGTTTTTCCTCTTGAGGTGAT 2137 human 2657 UUGUAUG TTTGTTGTATGTAAATGTTAA ACE2_ UGUAUUUGCUCAC 1938 GTCTCTTAAATCTTTTGTATTTGC 2138 human 3435 AGUGUUU TCACAGTGTTTGAGCAGTGCT ACE2_ AUUACUUGAACCA 1939 TCCACAAGAATGCTTATTACTTG 2139 human 643 GGUUUGA AACCAGGTTTGAATGAAATAAT ACE2_ AUGGCAAGAGCAA 1940 GTCTTGAAAAATGAGATGGCAA 2140 human 785 AUCAUUA GAGCAAATCATTATGAGGACTAT ACE2_ UGGACAGAAACCA 1941 TTTGACAGTTCCCTTTGGACAGA 2141 human 1072 AACAUAG AACCAAACATAGATGTTACTGA ACE2_ UGUAUUAGAGUAU 1942 TCTTAATAAGGATTTTGTATTAG 2142 human 2870 AUUAGGG AGTATATTAGGGAAAGTGTGTA ACE2_ AUAUCAUUGGAGC 1943 GTTGAAAACAAGGATATATCATT 2143 human 2974 AAGUGUU GGAGCAAGTGTTGGATCTTGTA ACE2_ GUAAAUGUUAAUU 1944 GTGATTTTGTTGTATGTAAATGT 2144 human 2676 UCAUGGU TAATTTCATGGTATAGAAAATA ACE2_ CUUAAUAAGGAUU 1945 ACTTCTGTTCTGTTTCTTAATAAG 2145 human 2856 UUGUAUU GATTTTGTATTAGAGTATATT ACE2_ CCCAAGUUCAAAG 1946 TTGAATAGCGCCCAACCCAAGTT 2146 human 122 GCUGAUA CAAAGGCTGATAAGAGAGAAAA ACE2_ UCUGCCAUUUACU 1947 CACGATTGTTGGGACTCTGCCAT 2147 human 1564 UACAUGU TTACTTACATGTTAGAGAAGTG ACE2_ AAGGCCACUGCUC 1948 AAAGAACATGAATGTAAGGCCA 2148 human 1960 AACUACU CTGCTCAACTACTTTGAGCCCTT ACE2_ GGGACGAUGUCAA 1949 GATCTTGGCTCACAGGGGACGAT 2149 human 212 GCUCUUC GTCAAGCTCTTCCTGGCTCCTT ACE2_ CACGAAGCCGAAG 1950 TTGGACAAGTTTAACCACGAAGC 2150 human 317 ACCUGUU CGAAGACCTGTTCTATCAAAGT ACE2_ ACAAGAAAUUCAG 1951 CCAAATGTATCCACTACAAGAA 2151 human 472 AAUCUCA ATTCAGAATCTCACAGTCAAGCT ACE2_ UGUGUCUGAUAUC 1952 CACTGCACCTAAAAATGTGTCTG 2152 human 2287 AUUCCUA ATATCATTCCTAGAACTGAAGT ACE2_ CAGGGAUAAUCUA 1953 GTCTCACAGGCTGTTCAGGGATA 2153 human 2919 AAUGUAA ATCTAAATGTAAATGTCTGTTG ACE2_ CUGAUAGAAACUC 1954 ACTCCCAGAGCATGCCTGATAGA 2154 human 3334 AUUUCUA AACTCATTTCTACTGTTCTCTA ACE2_ UAUGAUAUGGCAU 1955 ATGGGGCATATCCAGTATGATAT 2155 human 1358 AUGCUGC GGCATATGCTGCACAACCTTTT ACE2_ CACGAUUGUUGGG 1956 GCTCAAACAAGCACTCACGATTG 2156 human 1549 ACUCUGC TTGGGACTCTGCCATTTACTTA ACE2_ GAUGUUCAGACCU 1957 TTCCAAAACACTGATGATGTTCA 2157 human 2615 CCUUUUA GACCTCCTTTTAGAAAAATCTA ACE2_ AUAAGAUGAUAAA 1958 CATGGTATAGAAAATATAAGAT 2158 human 2705 GAUAUCA GATAAAGATATCATTAAATGTCA ACE2_ UGAUAAAGAUAUC 1959 ATAGAAAATATAAGATGATAAA 2159 human 2711 AUUAAAU GATATCATTAAATGTCAAAACTA ACE2_ GUUUCUUAAUAAG 1960 TTTGACTTCTGTTCTGTTTCTTAA 2160 human 2852 GAUUUUG TAAGGATTTTGTATTAGAGTA ACE2_ UUUGUAUUAGAGU 1961 TTTCTTAATAAGGATTTTGTATT 2161 human 2868 AUAUUAG AGAGTATATTAGGGAAAGTGTG ACE2_ CUUGGAAUUAUAA 1962 AAAGTTCACTTGCTTCTTGGAAT 2162 human 357 CACCAAU TATAACACCAATATTACTGAAG ACE2_ AGCCGUAUCAAUG 1963 ATCAGGATGTCCCGGAGCCGTAT 2163 human 2342 AUGCUUU CAATGATGCTTTCCGTCTGAAT ACE2_ AUUUCUGUCUCUG 1964 TTGCTTTCAGTATTTATTTCTGTC 2164 human 2822 GAUUUGA TCTGGATTTGACTTCTGTTCT ACE2_ GAGCACAAAGCAG 1965 TGTTTGAGCAGTGCTGAGCACAA 2165 human 3465 ACACUCA AGCAGACACTCAATAAATGCTA ACE2_ AAAGAUAUCAUUA 1966 AAAATATAAGATGATAAAGATA 2166 human 2715 AAUGUCA TCATTAAATGTCAAAACTATGAC ACE2_ GUGACCUUGACUG 1967 CTTTCTTACTTCCACGTGACCTTG 2167 human 90 AGUUUUG ACTGAGTTTTGAATAGCGCCC ACE2_ UGUGGAACAUACC 1968 CCAGTTGATTGAAGATGTGGAAC 2168 human 892 UUUGAAG ATACCTTTGAAGAGATTAAACC ACE2_ UAUGCUAUGAGGC 1969 CGATCATCTGTTGCATATGCTAT 2169 human 2162 AGUACUU GAGGCAGTACTTTTTAAAAGTA ACE2_ UUCCAUAUGGCUG 1970 TAACCAGCCCCCTGTTTCCATAT 2170 human 2434 AUUGUUU GGCTGATTGTTTTTGGAGTTGT ACE2_ ACUACAAUGAGAG 1971 TGGCAAACAGTTTAGACTACAAT 2171 human 687 GCUCUGG GAGAGGCTCTGGGCTTGGGAAA ACE2_ AUUUGAAACCAAG 1972 ATGTGCGAGTGGCTAATTTGAAA 2172 human 2238 AAUCUCC CCAAGAATCTCCTTTAATTTCT ACE2_ UGUUGAAUUUCUG 1973 CTAAATGTAAATGTCTGTTGAAT 2173 human 2944 AAGUUGA TTCTGAAGTTGAAAACAAGGAT ACE2_ CCCAGUCUCUUAA 1974 CCTCTGAAGTGGGTACCCAGTCT 2174 human 3416 AUCUUUU CTTAAATCTTTTGTATTTGCTC ACE2_ GAUGGGAGUGAUA 1975 TGTTTTTGGAGTTGTGATGGGAG 2175 human 2464 GUGGUUG TGATAGTGGTTGGCATTGTCAT ACE2_ UGAUGAUGUUCAG 1976 AGGATTCCAAAACACTGATGAT 2176 human 2611 ACCUCCU GTTCAGACCTCCTTTTAGAAAAA ACE2_ UGCUUCUUGGAAU 1977 CTATCAAAGTTCACTTGCTTCTT 2177 human 352 UAUAACA GGAATTATAACACCAATATTAC ACE2_ CUAAAUGUAAAUG 1978 CTGTTCAGGGATAATCTAAATGT 2178 human 2929 UCUGUUG AAATGTCTGTTGAATTTCTGAA ACE2_ AAUGCUGGGGACA 1979 GTCCAAAACATGAATAATGCTG 2179 human 407 AAUGGUC GGGACAAATGGTCTGCCTTTTTA ACE2_ UUUUCAAGAAGAC 1980 TCTTCTGTCACCCGATTTTCAAG 2180 human 1498 AAUGAAA AAGACAATGAAACAGAAATAAA ACE2_ UCUUGGAGAUAAA 1981 AAGCCTAAAATCAGCTCTTGGAG 2181 human 2098 GCAUAUG ATAAAGCATATGAATGGAACGA ACE2_ AAUGAAAUGUACC 1982 TATGAATGGAACGACAATGAAA 2182 human 2129 UGUUCCG TGTACCTGTTCCGATCATCTGTT ACE2_ AAUAUAAGAUGAU 1983 TTTCATGGTATAGAAAATATAAG 2183 human 2702 AAAGAUA ATGATAAAGATATCATTAAATG ACE2_ UGUUCUGUUUCUU 1984 TCTGGATTTGACTTCTGTTCTGTT 2184 human 2846 AAUAAGG TCTTAATAAGGATTTTGTATT ACE2_ UUUGCCUACAGUG 1985 CAAGTACTATGGTGATTTGCCTA 2185 human 3156 AUGUUUG CAGTGATGTTTGGAATCGATCA ACE2_ AAUCUCAUGAGGA 1986 GCTGATAAGAGAGAAAATCTCA 2186 human 150 GGUUUUA TGAGGAGGTTTTAGTCTAGGGAA ACE2_ UAAGAUGAUAAAG 1987 ATGGTATAGAAAATATAAGATG 2187 human 2706 AUAUCAU ATAAAGATATCATTAAATGTCAA ACE2_ GAUGUUUGGAAUC 1988 TGATTTGCCTACAGTGATGTTTG 2188 human 3168 GAUCAUG GAATCGATCATGCTTTCTTCAA ACE2_ CCACACUUGCCCA 1989 TTTTAAAGGAACAGTCCACACTT 2189 human 447 AAUGUAU GCCCAAATGTATCCACTACAAG ACE2_ GAGACUAUGAAGU 1990 GGGATTATTGGAGAGGAGACTA 2190 human 831 AAAUGGG TGAAGTAAATGGGGTAGATGGCT ACE2_ CUUCAUUGACAUU 1991 CCAAGGAGAGAGCATCTTCATTG 2191 human 2796 GCUUUCA ACATTGCTTTCAGTATTTATTT ACE2_ AAAUGUCUGUUGA 1992 GGATAATCTAAATGTAAATGTCT 2192 human 2937 AUUUCUG GTTGAATTTCTGAAGTTGAAAA ACE2_ UGUAGCUGCAAGG 1993 GTGCCTGGGAACTGGTGTAGCTG 2193 human 3049 AUUGAGA CAAGGATTGAGAATGGCATGCA ACE2_ GUUGACAUAGAUA 1994 TACAGAGGATCAGGAGTTGACA 2194 human 35 CUCUUUG TAGATACTCTTTGGATTTCATAC ACE2_ UGUCCAAAACAUG 1995 TATTACTGAAGAGAATGTCCAAA 2195 human 391 AAUAAUG ACATGAATAATGCTGGGGACAA ACE2_ GAUGAUAAAGAUA 1996 GTATAGAAAATATAAGATGATA 2196 human 2709 UCAUUAA AAGATATCATTAAATGTCAAAAC ACE2_ UUGUAUUAGAGUA 1997 TTCTTAATAAGGATTTTGTATTA 2197 human 2869 UAUUAGG GAGTATATTAGGGAAAGTGTGT ACE2_ UUCACAGUAACUC 1998 GGATGACATGCTTTCTTCACAGT 2198 human 3124 AGUUCAA AACTCAGTTCAAGTACTATGGT ACE2_ UCAGGAGUUGACA 1999 CATGGCTACAGAGGATCAGGAG 2199 human 29 UAGAUAC TTGACATAGATACTCTTTGGATT ACE2_ UGUACUCUUUGAC 2000 GATTTTGGACAAATCTGTACTCT 2200 human 1050 AGUUCCC TTGACAGTTCCCTTTGGACAGA ACE2_ GGAGCUAAUGAAG 2001 TTTCTGCTAAGAAATGGAGCTAA 2201 human 1400 GAUUCCA TGAAGGATTCCATGAAGCTGTT ACE2_ UGAGCCCUUAUUU 2002 ACTGCTCAACTACTTTGAGCCCT 2202 human 1981 ACCUGGC TATTTACCTGGCTGAAAGACCA ACE2_ UGCUAUGAGGCAG 2003 ATCATCTGTTGCATATGCTATGA 2203 human 2164 UACUUUU GGCAGTACTTTTTAAAAGTAAA ACE2_ GUAUGUAAAUGUU 2004 TGAGGTGATTTTGTTGTATGTAA 2204 human 2672 AAUUUCA ATGTTAATTTCATGGTATAGAA ACE2_ UCUUCAUUGACAU 2005 GCCAAGGAGAGAGCATCTTCATT 2205 human 2795 UGCUUUC GACATTGCTTTCAGTATTTATT ACE2_ UUUGGUCUCACAG 2006 TAGGGAAAGTGTGTATTTGGTCT 2206 human 2900 GCUGUUC CACAGGCTGTTCAGGGATAATC ACE2_ UAAGAGAGAAAAU 2007 AAGTTCAAAGGCTGATAAGAGA 2207 human 140 CUCAUGA GAAAATCTCATGAGGAGGTTTTA ACE2_ CAUGAGAUGGGGC 2008 TTCCTGACAGCTCATCATGAGAT 2208 human 1337 AUAUCCA GGGGCATATCCAGTATGATATG ACE2_ UUCUAAUGAUUAC 2009 ATCTCTGTTCCATGTTTCTAATG 2209 human 1735 UCAUUCA ATTACTCATTCATTCGATATTA ACE2_ GUAUCAAUGAUGC 2010 GGATGTCCCGGAGCCGTATCAAT 2210 human 2346 UUUCCGU GATGCTTTCCGTCTGAATGACA ACE2_ GUUGAAAACAAGG 2011 TGTTGAATTTCTGAAGTTGAAAA 2211 human 2959 AUAUAUC CAAGGATATATCATTGGAGCAA ACE2_ GAAGAUGUGGAAC 2012 CGCGGCCAGTTGATTGAAGATGT 2212 human 887 AUACCUU GGAACATACCTTTGAAGAGATT ACE2_ UUUGAAGAGAUUA 2013 GATGTGGAACATACCTTTGAAGA 2213 human 905 AACCAUU GATTAAACCATTATATGAACAT ACE2_ AGAACAAGAAUUC 2014 CCTGGCTGAAAGACCAGAACAA 2214 human 2010 UUUUGUG GAATTCTTTTGTGGGATGGAGTA ACE2_ UAGCAAAGGAGAA 2015 TGCCTCCATCGATATTAGCAAAG 2215 human 2575 AAUAAUC GAGAAAATAATCCAGGATTCCA ACE2_ GAAAACAAGGAUA 2016 TGAATTTCTGAAGTTGAAAACAA 2216 human 2962 UAUCAUU GGATATATCATTGGAGCAAGTG ACE2_ CAUGCUUUCUUCA 2017 ATGTTTGGAATCGATCATGCTTT 2217 human 3184 AGGUGAC CTTCAAGGTGACAGGTCTAAAG ACE2_ AACCCAAGUUCAA 2018 TTTTGAATAGCGCCCAACCCAAG 2218 human 120 AGGCUGA TTCAAAGGCTGATAAGAGAGAA ACE2_ AUGAACAUCUUCA 2019 AGATTAAACCATTATATGAACAT 2219 human 927 UGCCUAU CTTCATGCCTATGTGAGGGCAA ACE2_ ACUGGGAUCAGAG 2020 GTCATCCTGATCTTCACTGGGAT 2220 human 2504 AUCGGAA CAGAGATCGGAAGAAGAAAAAT ACE2_ UCUUCAAGGUGAC 2021 GAATCGATCATGCTTTCTTCAAG 2221 human 3191 AGGUCUA GTGACAGGTCTAAAGAGAGAAG ACE2_ UCAGCAAAAUGGG 2022 TCAGCTGCAGGCTCTTCAGCAAA 2222 human 517 UCUUCAG ATGGGTCTTCAGTGCTCTCAGA ACE2_ GCAAACAGUUUAG 2023 TTGAATGAAATAATGGCAAACA 2223 human 674 ACUACAA GTTTAGACTACAATGAGAGGCTC ACE2_ AAACCAUUAUAUG 2024 ACCTTTGAAGAGATTAAACCATT 2224 human 917 AACAUCU ATATGAACATCTTCATGCCTAT ACE2_ UCUGUACUCUUUG 2025 TAGATTTTGGACAAATCTGTACT 2225 human 1048 ACAGUUC CTTTGACAGTTCCCTTTGGACA ACE2_ UUUGACAGUUCCC 2026 GACAAATCTGTACTCTTTGACAG 2226 human 1057 UUUGGAC TTCCCTTTGGACAGAAACCAAA ACE2_ GAAACAGAAAUAA 2027 TTTCAAGAAGACAATGAAACAG 2227 human 1514 ACUUCCU AAATAAACTTCCTGCTCAAACAA ACE2_ CAGUAUUUAUUUC 2028 CATTGACATTGCTTTCAGTATTT 2228 human 2814 UGUCUCU ATTTCTGTCTCTGGATTTGACT ACE2_ CAGUUCAAGUACU 2029 TTCTTCACAGTAACTCAGTTCAA 2229 human 3136 AUGGUGA GTACTATGGTGATTTGCCTACA ACE2_ GUCCAAAACAUGA 2030 ATTACTGAAGAGAATGTCCAAA 2230 human 392 AUAAUGC ACATGAATAATGCTGGGGACAAA ACE2_ UUGAUGAAUGCCU 2031 TATGTGAGGGCAAAGTTGATGA 2231 human 959 AUCCUUC ATGCCTATCCTTCCTATATCAGT ACE2_ UGCAUAUGCUAUG 2032 GTTCCGATCATCTGTTGCATATG 2232 human 2158 AGGCAGU CTATGAGGCAGTACTTTTTAAA ACE2_ UAUGCCUCCAUCG 2033 AGTGGAGAAAATCCTTATGCCTC 2233 human 2558 AUAUUAG CATCGATATTAGCAAAGGAGAA ACE2_ AGUUGACAUAGAU 2034 CTACAGAGGATCAGGAGTTGAC 2234 human 34 ACUCUUU ATAGATACTCTTTGGATTTCATA ACE2_ UUUAGUCUAGGGA 2035 ATCTCATGAGGAGGTTTTAGTCT 2235 human 166 AAGUCAU AGGGAAAGTCATTCAGTGGATG ACE2_ UCCACACUUGCCC 2036 TTTTTAAAGGAACAGTCCACACT 2236 human 446 AAAUGUA TGCCCAAATGTATCCACTACAA ACE2_ UCUUCUGUCACCC 2037 TTTAAAATCCATTGGTCTTCTGT 2237 human 1483 GAUUUUC CACCCGATTTTCAAGAAGACAA ACE2_ AUAGAAAAUAUAA 2038 TGTTAATTTCATGGTATAGAAAA 2238 human 2696 GAUGAUA TATAAGATGATAAAGATATCAT ACE2_ UAUGCUGCACAAC 2039 CAGTATGATATGGCATATGCTGC 2239 human 1370 CUUUUCU ACAACCTTTTCTGCTAAGAAAT ACE2_ UGGAUUUGACUUC 2040 ATTTATTTCTGTCTCTGGATTTGA 2240 human 2833 UGUUCUG CTTCTGTTCTGTTTCTTAATA ACE2_ GACUUCUGUUCUG 2041 TCTGTCTCTGGATTTGACTTCTGT 2241 human 2840 UUUCUUA TCTGTTTCTTAATAAGGATTT ACE2_ CUUUCAUUUAAUC 2042 GCATGCATTAGCTCACTTTCATT 2242 human 3087 CAUUGUC TAATCCATTGTCAAGGATGACA ACE2_ AAGGAUGACAUGC 2043 ATTTAATCCATTGTCAAGGATGA 2243 human 3107 UUUCUUC CATGCTTTCTTCACAGTAACTC ACE2_ AGGUAGAGGACAU 2044 AAGAATCCAGGGAACAGGTAGA 2244 human 3233 UGCUUUU GGACATTGCTTTTTCACTTCCAA ACE2_ UUUGUAUCUGUUG 2045 GAGGCCGAGAAGTTCTTTGTATC 2245 human 1160 GUCUUCC TGTTGGTCTTCCTAATATGACT ACE2_ AUUCCCAAAGACC 2046 GTCTTTAAAGGGGAAATTCCCAA 2246 human 1619 AGUGGAU AGACCAGTGGATGAAAAAGTGG ACE2_ AAGACCAGAACAA 2047 TATTTACCTGGCTGAAAGACCAG 2247 human 2004 GAAUUCU AACAAGAATTCTTTTGTGGGAT ACE2_ UAAAGCAUAUGAA 2048 ATCAGCTCTTGGAGATAAAGCAT 2248 human 2107 UGGAACG ATGAATGGAACGACAATGAAAT ACE2_ CUGGAUUUGACUU 2049 TATTTATTTCTGTCTCTGGATTTG 2249 human 2832 CUGUUCU ACTTCTGTTCTGTTTCTTAAT ACE2_ UAGGGAAAGUGUG 2050 TGTATTAGAGTATATTAGGGAAA 2250 human 2885 UAUUUGG GTGTGTATTTGGTCTCACAGGC ACE2_ GCAUGCAUUAGCU 2051 CAAGGATTGAGAATGGCATGCA 2251 human 3072 CACUUUC TTAGCTCACTTTCATTTAATCCA ACE2_ UUGGUCUUCCUAA 2052 AGTTCTTTGTATCTGTTGGTCTTC 2252 human 1170 UAUGACU CTAATATGACTCAAGGATTCT ACE2_ GUUUCUAAUGAUU 2053 GCATCTCTGTTCCATGTTTCTAAT 2253 human 1733 ACUCAUU GATTACTCATTCATTCGATAT ACE2_ UUACACAAGGACC 2054 CTCATTCATTCGATATTACACAA 2254 human 1762 CUUUACC GGACCCTTTACCAATTCCAGTT ACE2_ UUGAAACCAAGAA 2055 GTGCGAGTGGCTAATTTGAAACC 2255 human 2240 UCUCCUU AAGAATCTCCTTTAATTTCTTT ACE2_ UCUGAUAUCAUUC 2056 GCACCTAAAAATGTGTCTGATAT 2256 human 2291 CUAGAAC CATTCCTAGAACTGAAGTTGAA ACE2_ AGUGCCUGGGAAC 2057 ATCACTTGTAAGGACAGTGCCTG 2257 human 3033 UGGUGUA GGAACTGGTGTAGCTGCAAGGA ACE2_ UGAGAAUGGCAUG 2058 TGTAGCTGCAAGGATTGAGAAT 2258 human 3064 CAUUAGC GGCATGCATTAGCTCACTTTCAT ACE2_ UGCAUUAGCUCAC 2059 GGATTGAGAATGGCATGCATTA 2259 human 3075 UUUCAUU GCTCACTTTCATTTAATCCATTG ACE2_ UCACCCUCUGAAG 2060 ATTCCAACTGTATGTTCACCCTC 2260 human 3397 UGGGUAC TGAAGTGGGTACCCAGTCTCTT ACE2_ UGAGGAGGUUUUA 2061 AGAGAGAAAATCTCATGAGGAG 2261 human 157 GUCUAGG GTTTTAGTCTAGGGAAAGTCATT ACE2_ UUUGCUUGGUGAU 2062 ATGCCTCCCTGCTCATTTGCTTG 2262 human 1012 AUGUGGG GTGATATGTGGGGTAGATTTTG ACE2_ UGGGGCAUAUCCA 2063 CAGCTCATCATGAGATGGGGCAT 2263 human 1344 GUAUGAU ATCCAGTATGATATGGCATATG ACE2_ AUAUGAAUGGAAC 2064 TCTTGGAGATAAAGCATATGAAT 2264 human 2113 GACAAUG GGAACGACAATGAAATGTACCT ACE2_ UUUCAUGGUAUAG 2065 GTATGTAAATGTTAATTTCATGG 2265 human 2687 AAAAUAU TATAGAAAATATAAGATGATAA ACE2_ UACUGAAGAGAAU 2066 TTATAACACCAATATTACTGAAG 2266 human 379 GUCCAAA AGAATGTCCAAAACATGAATAA ACE2_ GGCAAGAGCAAAU 2067 CTTGAAAAATGAGATGGCAAGA 2267 human 787 CAUUAUG GCAAATCATTATGAGGACTATGG ACE2_ UGCUGCACAACCU 2068 GTATGATATGGCATATGCTGCAC 2268 human 1372 UUUCUGC AACCTTTTCTGCTAAGAAATGG ACE2_ AAAGACCAGAACA 2069 TTATTTACCTGGCTGAAAGACCA 2269 human 2003 AGAAUUC GAACAAGAATTCTTTTGTGGGA ACE2_ AACGACAAUGAAA 2070 AAAGCATATGAATGGAACGACA 2270 human 2123 UGUACCU ATGAAATGTACCTGTTCCGATCA ACE2_ UGUGCGAGUGGCU 2071 TTTTGGGGAGGAGGATGTGCGA 2271 human 2224 AAUUUGA GTGGCTAATTTGAAACCAAGAAT ACE2_ UUAGCAAAGGAGA 2072 ATGCCTCCATCGATATTAGCAAA 2272 human 2574 AAAUAAU GGAGAAAATAATCCAGGATTCC ACE2_ GGAACAGGUAGAG 2073 GAGAGAAGAATCCAGGGAACAG 2273 human 3228 GACAUUG GTAGAGGACATTGCTTTTTCACT ACE2_ CUUAAAUCUUUUG 2074 GTGGGTACCCAGTCTCTTAAATC 2274 human 3424 UAUUUGC TTTTGTATTTGCTCACAGTGTT ACE2_ UAUCCACUACAAG 2075 ACACTTGCCCAAATGTATCCACT 2275 human 464 AAAUUCA ACAAGAAATTCAGAATCTCACA ACE2_ AAACCAAACAUAG 2076 GTTCCCTTTGGACAGAAACCAAA 2276 human 1079 AUGUUAC CATAGATGTTACTGATGCAATG ACE2_ CAAGGAUUCUGGG 2077 CTTCCTAATATGACTCAAGGATT 2277 human 1190 AAAAUUC CTGGGAAAATTCCATGCTAACG ACE2_ ACCAGAACAAGAA 2078 TTACCTGGCTGAAAGACCAGAA 2278 human 2007 UUCUUUU CAAGAATTCTTTTGTGGGATGGA ACE2_ AUGUUAAUUUCAU 2079 TTTTGTTGTATGTAAATGTTAATT 2279 human 2680 GGUAUAG TCATGGTATAGAAAATATAAG ACE2_ GAUAUCAUUAAAU 2080 ATATAAGATGATAAAGATATCAT 2280 human 2718 GUCAAAA TAAATGTCAAAACTATGACTCT ACE2_ UUAAAUGUCAAAA 2081 ATGATAAAGATATCATTAAATGT 2281 human 2725 CUAUGAC CAAAACTATGACTCTGTTCAGA ACE2_ CAGGCUGUUCAGG 2082 GTGTATTTGGTCTCACAGGCTGT 2282 human 2910 GAUAAUC TCAGGGATAATCTAAATGTAAA ACE2_ UGUCAAGGAUGAC 2083 TTTCATTTAATCCATTGTCAAGG 2283 human 3103 AUGCUUU ATGACATGCTTTCTTCACAGTA ACE2_ AUGGUGAUUUGCC 2084 CTCAGTTCAAGTACTATGGTGAT 2284 human 3149 UACAGUG TTGCCTACAGTGATGTTTGGAA ACE2_ UUGGACAAAUCUG 2085 TATGTGGGGTAGATTTTGGACAA 2285 human 1039 UACUCUU ATCTGTACTCTTTGACAGTTCC ACE2_ UCAAGAAGCACUU 2086 TTACCAATTCCAGTTTCAAGAAG 2286 human 1792 UGUCAAG CACTTTGTCAAGCAGCTAAACA ACE2_ UUUAAUUUCUUUG 2087 AAACCAAGAATCTCCTTTAATTT 2287 human 2258 UCACUGC CTTTGTCACTGCACCTAAAAAT ACE2_ GCAAGGAUUGAGA 2088 GGAACTGGTGTAGCTGCAAGGA 2288 human 3056 AUGGCAU TTGAGAATGGCATGCATTAGCTC ACE2_ UUGGAAUCGAUCA 2089 TGCCTACAGTGATGTTTGGAATC 2289 human 3173 UGCUUUC GATCATGCTTTCTTCAAGGTGA ACE2_ UCUCAUGAGGAGG 2090 TGATAAGAGAGAAAATCTCATG 2290 human 152 UUUUAGU AGGAGGTTTTAGTCTAGGGAAAG ACE2_ CAUUAUAUGAAGA 2091 GCAAGCAGCTGAGGCCATTATAT 2291 human 750 GUAUGUG GAAGAGTATGTGGTCTTGAAAA ACE2_ UCUGGGAAAAUUC 2092 ATATGACTCAAGGATTCTGGGAA 2292 human 1197 CAUGCUA AATTCCATGCTAACGGACCCAG ACE2_ UUCAGGAUCCUUA 2093 CTGGGGAAGGGCGACTTCAGGA 2293 human 1283 UGUGCAC TCCTTATGTGCACAAAGGTGACA ACE2_ AGGCCCUCUGCAC 2094 AGCAGCTAAACATGAAGGCCCT 2294 human 1825 AAAUGUG CTGCACAAATGTGACATCTCAAA

TABLE 12A Host targets screened-TMPRSS-20 nucleotide targets and 45 nucleotide gene target regions SEQ SEQ Sequence ID ID ID Sequence NO: Gene Region NO: Species TMPRSS UGUGAAAAUGA 2295 ACTGTAAAGTTCAATTGTGAAAAT 2331 human 2_3153 AUAUCAUGC GAATATCATGCAAATAAATTA TMPRSS ACCUUCAUUUA 2296 GTCTCCAAGTAGTCCACCTTCATT 2332 human 2_2577 ACUCUUUGA TAACTCTTTGAAACTGTATCA TMPRSS UCGUCCUUGACG 2297 TAATCCACATGGTCTTCGTCCTTG 2333 human 2_1626 UCGUUUUA ACGTCGTTTTACAAGAAAACA TMPRSS GGAGCCGGAUA 2298 TCTTTCATGTTCTATGGAGCCGGA 2334 human 2_1101 CCAAGUAGA TACCAAGTAGAAAAAGTGATT TMPRSS UACCACAGUGA 2299 ATCTATAAAAAACTGTACCACAGT 2335 human 2_810 UGCCUGUUC GATGCCTGTTCTTCAAAAGCA TMPRSS UCAUGCAAAUA 2300 TTGTGAAAATGAATATCATGCAAA 2336 human 2_3167 AAUUAUGCA TAAATTATGCAATTTTTTTTT TMPRSS UUGAAACUGUA 2301 CCTTCATTTAACTCTTTGAAACTGT 2337 human 2_593 UCAUCUUUG ATCATCTTTGCCAAGTAAGA TMPRSS GCCGGCAAUGUC 2302 AAACTGAACACAAGTGCCGGCAA 2338 human 2_780 GAUAUCUA TGTCGATATCTATAAAAAACTG TMPRSS UGUAAUGGUGA 2303 CATCCTAAAAGGTGTTGTAATGGT 2339 human 2_3054 AAACGUCUU GAAAACGTCTTCCTTCTTTAT TMPRSS GGUGGCCUAUU 2304 TTGCCAAGTAAGAGTGGTGGCCTA 2340 human 2_2625 UCAGCUGCU TTTCAGCTGCTTTGACAAAAT TMPRSS ACAGCUAGGAC 2305 GAAATGAATGATTCTACAGCTAGG 2341 human 2_2899 UUAACCUUG ACTTAACCTTGAAATGGAAAG TMPRSS CAGUUUAAGGU 2306 GAAATCAAGGATGCTCAGTTTAAG 2342 human 2_2485 ACACUGUUU GTACACTGTTTCCATGTTATG TMPRSS GCCGCCAGAGCA 2307 TCAACTTGAACTCAAGCCGCCAGA 2343 human 2_886 GGAUUGUG GCAGGATTGTGGGCGGCGAGA TMPRSS CCAGCCAUGAUC 2308 GACAACCTGATCACACCAGCCATG 2344 human 2_1398 UGUGCCGG ATCTGTGCCGGCTTCCTGCAG TMPRSS UCCAUCAUCACC 2309 CACGTGTGCGGAGGCTCCATCATC 2345 human 2_984 CCCGAGUG ACCCCCGAGTGGATCGTGACA TMPRSS AUGAUCUGUGC 2310 CTGATCACACCAGCCATGATCTGT 2346 human 2_1404 CGGCUUCCU GCCGGCTTCCTGCAGGGGAAC TMPRSS GCUUUGAACUC 2311 GATAACAGCAAGATGGCTTTGAA 2347 human 2_138 AGGGUCACC CTCAGGGTCACCACCAGCTATT TMPRSS AGGAGAAAGGG 2312 GGTGGGGGGCCACCGAGGAGAAA 2348 human 2_1297 AAGACCUCA GGGAAGACCTCAGAAGTGCTGA TMPRSS CCUGGCAGGUCA 2313 TCCCGGGGGCCTGGCCCTGGCAGG 2349 human 2_937 GCCUGCAC TCAGCCTGCACGTCCAGAACG TMPRSS GAGGAGAAAGG 2314 GGGTGGGGGGCCACCGAGGAGAA 2350 human 2_1296 GAAGACCUC AGGGAAGACCTCAGAAGTGCTG TMPRSS CAGGUCAGCCUG 2315 GGGGCCTGGCCCTGGCAGGTCAG 2351 human 2_942 CACGUCCA CCTGCACGTCCAGAACGTCCAC TMPRSS CAUUGGACGGC 2316 CTTAACAATCCATGGCATTGGACG 2352 human 2_1053 AUUUGCGGG GCATTTGCGGGGATTTTGAGA TMPRSS GAGGCUCCAUCA 2317 ACGTCCACGTGTGCGGAGGCTCCA 2353 human 2_979 UCACCCCC TCATCACCCCCGAGTGGATCG TMPRSS CAUGAUCUGUG 2318 CCTGATCACACCAGCCATGATCTG 2354 human 2_1403 CCGGCUUCC TGCCGGCTTCCTGCAGGGGAA TMPRSS ACCAGCCAUGAU 2319 TGACAACCTGATCACACCAGCCAT 2355 human 2_1397 CUGUGCCG GATCTGTGCCGGCTTCCTGCA TMPRSS AAAGCCAUGCCA 962 TGGGTTTATACCAGGAAAGCCATG 1162 mouse 2_2779 GAAUUACC CCAGAATTACCAAATATGAAG TMPRSS UUUGUCUUCAA 2320 TTGTCCCAGACTTCCTTTGTCTTCA 2356 mouse 2_1730 CAACCUUCU ACAACCTTCTGCAAGAAAAC TMPRSS UGCACAAUGUA 2321 AATTTTAACTTCCTGTGCACAATG 2357 mouse 2_1785 CCUUUUGAG TACCTTTTGAGATGATTCGAA TMPRSS UGGGACAGCAA 2322 TTGCTTTGGAGGTTCTGGGACAGC 2358 mouse 2_552 CUGUUCUAC AACTGTTCTACGTCTGAGATG TMPRSS UUCCACUGUGA 2323 TTCTGAGCTGTGAGATTCCACTGT 2359 mouse 2_3120 AAUAUAUGA GAAATATATGAATAAAGTATA TMPRSS UCAGGCAACGU 2324 AAGCTGAATGTGAGCTCAGGCAA 2360 mouse 2_873 UGACCUCUA CGTTGACCTCTATAAAAAACTC TMPRSS GGGAACGUGAC 2325 AGACCTGGAGTATACGGGAACGT 2361 mouse 2_1647 GGUAUUUAC GACGGTATTTACAGATTGGATC TMPRSS UCUGCAAGAAA 2326 TGTCTTCAACAACCTTCTGCAAGA 2362 mouse 2_1747 ACCAAGGGC AAACCAAGGGCCTGAATTTTA TMPRSS UUUGGCUUUUA 2327 GAAGCTGCAGACACCTTTGGCTTT 2363 mouse 2_1286 AUGAUCUAG TAATGATCTAGTGAAGCCAGT TMPRSS UAAGCGAGAAC 2328 CCGCCTCCGGAGATTTAAGCGAGA 2364 mouse 2_88 UGGAGUAGG ACTGGAGTAGGTCGTGTACTT TMPRSS GUUGACAUGAC 2329 CTTGCTCTCCTGCATGTTGACATG 2365 mouse 2_2243 GGCCCUUUC ACGGCCCTTTCCAAGGGTGAT TMPRSS UGCUUCUGGGU 2330 TGATTTCAGTCACCTTGCTTCTGG 2366 mouse 2_2539 UGUGUUUCU GTTGTGTTTCTTCTCTTACTA

TABLE 12B Host targets screened - IL-6-20 nucleotide targets and 45 nucleotide gene target regions Se-  SEQ SEQ quence ID ID ID Sequence NO: Gene Region NO: Species IL6_ UUGGAAAGUGUA 2367 GAGCCAGATCATTTCTTGGAAAG 2389 human 933 GGCUUACC TGTAGGCTTACCTCAAATAAAT IL6_ CUUGAAAUGUUA 2368 TTTTATGAAGTGTCACTTGAAAT 2390 mouse 930 UAUGUUAU GTTATATGTTATAGTTTTGAAA IL6_ UAUGAUUGAUAU 2369 AACTTTAAGTTAATTTATGATTG 2391 mouse 896 UUAUUAUU ATATTTATTATTTTTATGAAGT IL6_ UUAAAUAAGUAA 2370 TAATTTATTGATAATTTAAATAA 2392 mouse 870 ACUUUAAG GTAAACTTTAAGTTAATTTATG IL6_ GACACUAUUUUA 2371 CAATATGAATGTTGGGACACTAT 2393 mouse 835 AUUAUUUU TTTAATTATTTTTAATTTATTG IL6_ GGACACUAUUUU 2372 ACAATATGAATGTTGGGACACTA 2394 mouse 834 AAUUAUUU TTTTAATTATTTTTAATTTATT IL6_ UGGGACACUAUU 2373 TGACAATATGAATGTTGGGACAC 2395 mouse 832 UUAAUUAU TATTTTAATTATTTTTAATTTA IL6_ GUGGACAUUCCUC 2374 TAAGCATATCAGTTTGTGGACAT 2396 mouse 741 ACUGUGG TCCTCACTGTGGTCAGAAAATA IL6_ UGAAGAAUUUCU 2375 CATCTTGAAATCACTTGAAGAAT 2397 mouse 669 AAAAGUCA TTCTAAAAGTCACTTTGAGATC IL6_ AUGGGCACCUCAG 2376 TTCGGCAAATGTAGCATGGGCAC 2398 human 635 AUUGUUG CTCAGATTGTTGTTGTTAATGG IL6_ UCGGCAAAUGUA 2377 CAGCCTGAGGGCTCTTCGGCAAA 2399 human 621 GCAUGGGC TGTAGCATGGGCACCTCAGATT IL6_ UCGGCAAAUGUA 2377 CAGCCTGAGGGCTCTTCGGCAAA 2399 human 621 GCAUGGGC TGTAGCATGGGCACCTCAGATT IL6_ GAUAUAAUCAGG 2378 GATGCTACCAAACTGGATATAAT 2400 mouse 386 AAAUUUGC CAGGAAATTTGCCTATTGAAAA IL6_ UGAGGUAUACCU 2379 TGGTCTTTTGGAGTTTGAGGTAT 2401 human 360 AGAGUACC ACCTAGAGTACCTCCAGAACAG IL6_ GGUGAAAAUCAU 2380 TGAGGAGACTTGCCTGGTGAAAA 2402 human 330 CACUGGUC TCATCACTGGTCTTTTGGAGTT IL6_ GGUGAAAAUCAU 2380 TGAGGAGACTTGCCTGGTGAAAA 2402 human 330 CACUGGUC TCATCACTGGTCTTTTGGAGTT IL6_ CUUGCCUGGUGAA 2381 GATTCAATGAGGAGACTTGCCTG 2403 human 323 AAUCAUC GTGAAAATCATCACTGGTCTTT IL6_ CUUGCCUGGUGAA 2381 GATTCAATGAGGAGACTTGCCTG 2403 human 323 AAUCAUC GTGAAAATCATCACTGGTCTTT IL6_ UGUGCAAUGGCA 2382 AAATGAGAAAAGAGTTGTGCAA 2404 mouse 284 AUUCUGAU TGGCAATTCTGATTGTATGAACA IL6_ UGAACCUUCCAAA 2383 TGGCAGAAAACAACCTGAACCTT 2405 human 263 GAUGGCU CCAAAGATGGCTGAAAAAGATG IL6_ ACUGGCAGAAAAC 2384 AAGCAGCAAAGAGGCACTGGCA 2406 human 246 AACCUGA GAAAACAACCTGAACCTTCCAAA IL6_ ACUGGCAGAAAAC 2384 AAGCAGCAAAGAGGCACTGGCA 2406 human 246 AACCUGA GAAAACAACCTGAACCTTCCAAA IL6_ GAAAGCAGCAAA 2385 AAGAGTAACATGTGTGAAAGCA 2407 human 229 GAGGCACU GCAAAGAGGCACTGGCAGAAAAC IL6_ GAAAGCAGCAAA 2385 AAGAGTAACATGTGTGAAAGCA 2407 human 229 GAGGCACU GCAAAGAGGCACTGGCAGAAAAC IL6_ UCAGCCCUGAGAA 2386 ATCCTCGACGGCATCTCAGCCCT 2408 human 187 AGGAGAC GAGAAAGGAGACATGTAACAAG IL6_ UGCUAAUUUAAA 2387 TTTACCTCAATGAATTGCTAATTT 2409 mouse 1045 UAUGUUUU AAATATGTTTTTAAAGAAATC IL6_ CUUGGAAUGUAU 2388 CTAGCCAGATGGTTTCTTGGAAT 2410 mouse 1015 AAGUUUAC GTATAAGTTTACCTCAATGAAT

TABLE 12C Host targets screened - ACE2_-20 nucleotide targets and 45 nucleotide gene target regions Se- SEQ SEQ quence ID ID ID Sequence NO: Gene Region NO: Species ACE2_ CAACCCAAGUUC 2411 GTTTTGAATAGCGCCCAACCCAAG 2435 human 119 AAAGGCUG TTCAAAGGCTGATAAGAGAGA ACE2_ UCUAUCAAAGUU 2412 AAGCCGAAGACCTGTTCTATCAAA 2436 human 336 CACUUGCU GTTCACTTGCTTCTTGGAATT ACE2_ ACUUGCUUCUUG 2413 GTTCTATCAAAGTTCACTTGCTTCT 2437 human 349 GAAUUAUA TGGAATTATAACACCAATAT ACE2_ UUGGAAUUAUAA 2414 AAGTTCACTTGCTTCTTGGAATTA 2438 human 358 CACCAAUA TAACACCAATATTACTGAAGA ACE2_ AAUCCACAAGAA 2415 GTTTGTAACCCAGATAATCCACAA 2439 human 626 UGCUUAUU GAATGCTTATTACTTGAACCA ACE2_ GAUGGCAAGAGC 2416 GGTCTTGAAAAATGAGATGGCAA 2440 human 784 AAAUCAUU GAGCAAATCATTATGAGGACTA ACE2_ GAAGAGAUUAAA 2417 GTGGAACATACCTTTGAAGAGATT 2441 human 908 CCAUUAUA AAACCATTATATGAACATCTT ACE2_ AGAUUUUGGACA 2418 GGTGATATGTGGGGTAGATTTTGG 2442 human 1034 AAUCUGUA ACAAATCTGTACTCTTTGACA ACE2_ UUGGACAGAAAC 2419 CTTTGACAGTTCCCTTTGGACAGA 2443 human 1071 CAAACAUA AACCAAACATAGATGTTACTG ACE2_ CCUAAUAUGACU 2420 GTATCTGTTGGTCTTCCTAATATG 2444 human 1178 CAAGGAUU ACTCAAGGATTCTGGGAAAAT ACE2_ UCUGCUAAGAAA 2421 TGCTGCACAACCTTTTCTGCTAAG 2445 human 1387 UGGAGCUA AAATGGAGCTAATGAAGGATT ACE2_ GGGGAAAUCAUG 2422 TTCCATGAAGCTGTTGGGGAAATC 2446 human 1430 UCACUUUC ATGTCACTTTCTGCAGCCACA ACE2_ UGAAACAGAAAU 2423 TTTTCAAGAAGACAATGAAACAG 2447 human 1513 AAACUUCC AAATAAACTTCCTGCTCAAACA ACE2_ UCACGAUUGUUG 2424 TGCTCAAACAAGCACTCACGATTG 2448 human 1548 GGACUCUG TTGGGACTCTGCCATTTACTT ACE2_ GUGGAGGUGGAU 2425 TTACATGTTAGAGAAGTGGAGGTG 2449 human 1591 GGUCUUUA GATGGTCTTTAAAGGGGAAAT ACE2_ CUAAUGAUUACU 2426 CTCTGTTCCATGTTTCTAATGATTA 2450 human 1737 CAUUCAUU CTCATTCATTCGATATTACA ACE2_ UUCGAUAUUACA 2427 ATGATTACTCATTCATTCGATATT 2451 human 1755 CAAGGACC ACACAAGGACCCTTTACCAAT ACE2_ CUUUACCAAUUC 2428 TATTACACAAGGACCCTTTACCAA 2452 human 1775 CAGUUUCA TTCCAGTTTCAAGAAGCACTT ACE2_ CUGCACAAAUGU 2429 AAACATGAAGGCCCTCTGCACAA 2453 human 1832 GACAUCUC ATGTGACATCTCAAACTCTACA ACE2_ UAGAAAAUAUAA 2430 GTTAATTTCATGGTATAGAAAATA 2454 human 2697 GAUGAUAA TAAGATGATAAAGATATCATT ACE2_ AUGGCCAAGGAG 2431 TTGTCCAAAGACAACATGGCCAAG 2455 human 2777 AGAGCAUC GAGAGAGCATCTTCATTGACA ACE2_ AUUGACAUUGCU 2432 GGAGAGAGCATCTTCATTGACATT 2456 human 2800 UUCAGUAU GCTTTCAGTATTTATTTCTGT ACE2_ UUCAGUAUUUAU 2433 TTCATTGACATTGCTTTCAGTATTT 2457 human 2812 UUCUGUCU ATTTCTGTCTCTGGATTTGA ACE2_ UUUGGAAUCGAU 2434 TTGCCTACAGTGATGTTTGGAATC 2458 human 3172 CAUGCUUU GATCATGCTTTCTTCAAGGTG

TABLE 12D Host targets screened - FURIN-20 nucleotide targets and 45 nucleotide gene target regions Se- SEQ SEQ quence  ID ID ID Sequence NO: Gene Region NO: Species FURIN_ UCAACCUGGG 2459 GGAAGCATGGGTTCCTCAACCTGG 2483 human 443 CCAGAUCUUC GCCAGATCTTCGGGGACTATT FURIN_ AUUACCACUU 2460 AGATCTTCGGGGACTATTACCACTT 2484 human 470 CUGGCAUCGA CTGGCATCGAGGAGTGACGA FURIN_ GCAGGCAAUU 2461 AACCACCCGGACTTGGCAGGCAAT 2485 human 769 AUGAUCCUGG TATGATCCTGGGGCCAGTTTT FURIN_ UUUGAUGUCA 2462 GATCCTGGGGCCAGTTTTGATGTCA 2486 human 796 AUGACCAGGA ATGACCAGGACCCTGACCCC FURIN_ GAGGCCAAUA 2463 ATTGCTCTCACCCTGGAGGCCAAT 2487 human 1426 AGAACCUCAC AAGAACCTCACATGGCGGGAC FURIN_ UUCAUGACAA 2464 TTTAATGACTGGGCCTTCATGACAA 2488 human 1876 CUCAUUCCUG CTCATTCCTGGGATGAGGAT FURIN_ UGGGACGCUG 2465 CGAAGCCAACAACTATGGGACGCT 2489 human 1959 ACCAAGUUCA GACCAAGTTCACCCTCGTACT FURIN_ UGACCAAGUU 2466 ACAACTATGGGACGCTGACCAAGT 2490 human 1967 CACCCUCGUA TCACCCTCGTACTCTATGGCA FURIN_ UUUUAAUUCA 2467 CCCTCCTTGGGCACTTTTTAATTCA 2491 human 2711 CCAAAGUAUU CCAAAGTATTTTTTTATCTT FURIN_ UUUAAUUCAC 2468 CCTCCTTGGGCACTTTTTAATTCAC 2492 human 2712 CAAAGUAUUU CAAAGTATTTTTTTATCTTG FURIN_ UGUUUGAGGA 2469 GGGGATCTCAGGGGCTGTTTGAGG 2493 human 3524 UAUAUUUUCA ATATATTTTCACTTTGTGATT FURIN_ UUUGAGGAUA 2470 GGATCTCAGGGGCTGTTTGAGGAT 2494 human 3526 UAUUUUCACU ATATTTTCACTTTGTGATTAT FURIN_ UGAGGAUAUA 2471 ATCTCAGGGGCTGTTTGAGGATAT 2495 human 3528 UUUUCACUUU ATTTTCACTTTGTGATTATTT FURIN_ UUUCACUUUG 2472 TGTTTGAGGATATATTTTCACTTTG 2496 human 3539 UGAUUAUUUC TGATTATTTCACTTTAGATG FURIN_ UAUUUCACUU 2473 TTTTCACTTTGTGATTATTTCACTTT 2497 human 3553 UAGAUGCUGA AGATGCTGATGATTTGTTT FURIN_ UUAGAUGCUG 2474 TGTGATTATTTCACTTTAGATGCTG 2498 human 3562 AUGAUUUGUU ATGATTTGTTTTTGTATTTT FURIN_ CUGGUUUUGU 2475 TCGTGGCCAGCCCGGCTGGTTTTGT 2499 human 4101 AAGAUGCUGG AAGATGCTGGGTTGGTGCAC FURIN_ UGGGUUGGUG 2476 GGTTTTGTAAGATGCTGGGTTGGTG 2500 human 4118 CACAGUGAUU CACAGTGATTTTTTTCTTGT FURIN_ CUUGUAAUUU 2477 CACAGTGATTTTTTTCTTGTAATTT 2501 human 4143 AAACAGGCCC AAACAGGCCCAGCATTGCTG FURIN_ UUAAACAGGC 2478 TTTTTTTCTTGTAATTTAAACAGGC 2502 human 4151 CCAGCAUUGC CCAGCATTGCTGGTTCTATT FURIN_ CCCAGCAUUG 2479 TGTAATTTAAACAGGCCCAGCATT 2503 human 4160 CUGGUUCUAU GCTGGTTCTATTTAATGGACA FURIN_ UGAGAUAAUG 2480 TCTATTTAATGGACATGAGATAAT 2504 human 4190 UUAGAGGUUU GTTAGAGGTTTTAAAGTGATT FURIN_ GAGAUAAUGU 2481 CTATTTAATGGACATGAGATAATG 2505 human 4191 UAGAGGUUUU TTAGAGGTTTTAAAGTGATTA FURIN_ UUAGAGGUUU 2482 GGACATGAGATAATGTTAGAGGTT 2506 human 4200 UAAAGUGAUU TTAAAGTGATTAAACGTGCAG

TABLE 12E Host targets screened - IL-6R-20 nucleotide targets and 45 nucleotide  gene target regions Se- SEQ SEQ quence ID ID ID Sequence NO: Gene Region NO: Species IL6R_ UGAGUCAUGUG 2507 GACCGTCCGCCGCTCTGAGTCAT 2529 human 38 CGAGUGGGA GTGCGAGTGGGAAGTCGCACTG IL6R_ AGAGCCGGAAG 2508 GACCTGCCCGGGGGTAGAGCCG 2530 human 437 ACAAUGCCA GAAGACAATGCCACTGTTCACTG IL6R_ CUCAGCAAUGU 2509 TTCCGGAAGAGCCCCCTCAGCAA 2531 human 666 UGUUUGUGA TGTTGTTTGTGAGTGGGGTCCT IL6R_ GCUCUUGGUGA 2510 GACGACAAAGGCTGTGCTCTTGG 2532 human 728 GGAAGUUUC TGAGGAAGTTTCAGAACAGTCC IL6R_ AGUGUCGGGAG 2511 ATGTGCGTCGCCAGTAGTGTCGG 2533 human 876 CAAGUUCAG GAGCAAGTTCAGCAAAACTCAA IL6R_ UUCAGCAAAAC 2512 AGTGTCGGGAGCAAGTTCAGCA 2534 human 891 UCAAACCUU AAACTCAAACCTTTCAGGGTTGT IL6R_ UCAAACCUUUCA 2513 CAAGTTCAGCAAAACTCAAACCT 2535 human 902 GGGUUGUG TTCAGGGTTGTGGAATCTTGCA IL6R_ UUCAGGGUUGU 2514 GCAAAACTCAAACCTTTCAGGGT 2536 human 910 GGAAUCUUG TGTGGAATCTTGCAGCCTGATC IL6R_ CACUCCUGGAAC 2515 ACCTGGCAAGACCCCCACTCCTG 2537 human 1011 UCAUCUUU GAACTCATCTTTCTACAGACTA IL6R_ ACUCCUGGAACU 2516 CCTGGCAAGACCCCCACTCCTGG 2538 human 1012 CAUCUUUC AACTCATCTTTCTACAGACTAC IL6R_ GGUUUGAGCUC 2517 CTTTCTACAGACTACGGTTTGAG 2539 human 1042 AGAUAUCGG CTCAGATATCGGGCTGAACGGT IL6R_ UCAGAUAUCGG 2518 GACTACGGTTTGAGCTCAGATAT 2540 human 1051 GCUGAACGG CGGGCTGAACGGTCAAAGACAT IL6R_ CUGAACGGUCA 2519 AGCTCAGATATCGGGCTGAACG 2541 human 1063 AAGACAUUC GTCAAAGACATTCACAACATGGA IL6R_ AAAGACAUUCA 2520 TCGGGCTGAACGGTCAAAGACA 2542 human 1073 CAACAUGGA TTCACAACATGGATGGTCAAGGA IL6R_ UCCAGCAUCACU 2521 GGATGGTCAAGGACCTCCAGCA 2543 human 1105 GUGUCAUC TCACTGTGTCATCCACGACGCCT IL6R_ UUGGACAGAAG 2522 GGCCATGGGCACGCCTTGGACA 2544 human 1229 GUCUCCUGA GAAGGTCTCCTGAGAGGGTCACT IL6R_ CCUGAGAGGGU 2523 TTGGACAGAAGGTCTCCTGAGA 2545 human 1244 CACUGCAAA GGGTCACTGCAAAAGAGAATCTC IL6R_ CACUGCAAAAG 2524 GTCTCCTGAGAGGGTCACTGCAA 2546 human 1255 AGAAUCUCG AAGAGAATCTCGTTCCAACCTC IL6R_ UGCAAAAGAGA 2525 TCCTGAGAGGGTCACTGCAAAA 2547 human 1258 AUCUCGUUC GAGAATCTCGTTCCAACCTCCCT IL6R_ GUGGACCACGCC 2526 CCTGTCAATCTGAACGTGGACCA 2548 human 1350 UAAACUAA CGCCTAAACTAATTTTTGACTG IL6R_ UGGACCACGCCU 2527 CTGTCAATCTGAACGTGGACCAC 2549 human 1351 AAACUAAU GCCTAAACTAATTTTTGACTGC IL6R_ UGUGCCAGCUG 2528 CTAATTTTTGACTGCTGTGCCAG 2550 human 1381 GAGUGAUGA CTGGAGTGATGATAGGCTCACT

Using a novel algorithm, a panel of siRNAs targeting various regions of ACE2 and FURIN mRNA were designed (FIG. 12 ). For ASOs, a second step of selection involved testing the secondary structure (accessibility) of the target using the online algorithm lncASO. The sequences of ASOs targeting ACE2 and FURIN are summarized in Table 13A and Table 131B, respectively.

TABLE 13A ASOs targeting host factors-ACE2 target SEQ  homology Sequence ID  Mon- Oligo ID (anti sense) NO: Human Mouse key ACE2_171 GCAAGTGAACTTTGAT 2551 Y Y Y ACE2_250 AAAGGCAGACCATTTG 2552 Y Y N ACE2_567 GGCCTCAGCTGCTTGC 2553 Y Y Y ACE2_694 GCCGCGGCTGTAGTCA 2554 Y N N ACE2_702 ATCAACTGGCCGCGGC 2555 Y N N ACE2_851 TACCCCACATATCACC 2556 Y Y Y ACE2_938 AGGCCTGGTCCACCAT 2557 Y N N ACE2_1326 TCTTCTTGAAAATCGG 2558 Y Y N ACE2_1425 AAGACCATCCACCTCC 2559 Y Y Y ACE2_1533 GGGTCACAGTATGTTT 2560 Y Y Y ACE2_1666 GATGTCACATTTGTGC 2561 Y Y Y ACE2_2806 GAGTTCACGGAGGCCC 2562 Y N N

TABLE 13B ASOs targeting host factors-FURIN target SEQ homology Sequence ID  Mon- Oligo ID (anti sense) NO: Human Mouse key FURIN_176 CCGGGGCTGACTGGTG 2563 Y Y Y FURIN_450 AAGATCTGGCCCAGGT 2564 Y Y N FURIN_963 GTCACCTCGCCATCCA 2565 Y Y Y FURIN_1044 TCATCCTCGGGGCCCC 2566 Y N Y FURIN_1184 CGCAGTTGCAGCTGTC 2567 Y N Y FURIN_1229 TGGCGCTGCTGATGGA 2568 Y Y Y FURIN_1400 TGATGCCGGCTGCTAA 2569 Y N Y FURIN_1610 GCTGGGGGGCCACTGT 2570 Y Y N FURIN_2213 CACATGAGGCGTGGCA 2571 Y Y Y FURIN_2217 GTGGCACATGAGGCGT 2572 Y Y Y FURIN_2649 AGGGCGCTCTGGTCTT 2573 Y Y Y FURIN_2653 TCAGAGGGCGCTCTGG 2574 Y N Y

SiRNAs targeting ACE2 and FURIN, two endogenous genes necessary for viral entry and spread were tested for silencing efficacy. FIG. 13A-13B depict the identification of siRNA hits for ACE2 and FURIN, respectively. For each target, at least 3 siRNAs were identified that reduced target mRNA expression below 75% compared to untreated controls. siRNAs were tested in human Hacat cells and silencing was assessed using the QuantiGene assay and confirmed using psicheck reporter system. FIG. 14A-14B depict validation and determination of IC₅₀ values for siRNAs targeting ACE2 (FIG. 14A) and FURIN (FIG. 14B). SiRNAs targeting ACE2 and FURIN were tested for silencing efficacy in 8-point dose response studies. Each siRNA showed potent and efficacious target silencing with IC₅₀ values in the low nanomolar range. FIG. 15A-15D depict validation and determination of IC₅₀ values for four selected siRNAs, tested for silencing efficacy in 8-point dose response studies. Each siRNA showed potent and efficacious target silencing with IC₅₀ values in the low nanomolar range. siRNAs were tested in HaCat cells and silencing was assessed using QuantiGene.

The identification of ASO hits for ACE2 and FURIN are shown in FIG. 16 . Twelve LNA gapmers targeting ACE2 (FIG. 16A) and FURIN (FIG. 16B), two endogenous genes necessary for viral entry and spread, were tested for silencing efficacy. For each target, at least 3 ASOs were identified that reduced target mRNA expression below 75% compared to untreated controls. ASOs were tested in human U2OS cells and silencing was assessed using QRT-PCR assay. FIG. 17A-17B depict the identification of ASO hits for ACE2 and FURIN. Twelve LNA gapmers targeting ACE2 (FIG. 17A) and FURIN (FIG. 17B), two endogenous genes necessary for viral entry and spread for silencing efficacy. For each target, we identified at least 3 ASOs that reduced target mRNA expression below 75% compared to untreated controls. ASOs were tested in human U2OS cells and silencing was assessed using QRT-PCR assay. Concentration: 1.5 μM; Time point: 72 hours. FIG. 18 depict validation and determination of IC₅₀ values for ASOs targeting ACE2 (FIG. 18A) and FURIN (FIG. 18B). ASOs targeting ACE2 and FURIN were tested for silencing efficacy in 3-point dose response and gene expression was measured using QRT-PCR. Concentration: Top=1.5 μM; Time point: 5 days.

Example 3. Delivery to the Lungs

In the present invention, methodologies are disclosed for a uniform and efficient delivery of fully stabilized siRNAs to the lung. Lung delivery was achieved after intratracheal administration (IT). This route might have significant advantages when using siRNA cocktails for prophylaxis and in the field as it is minimally invasive. Efficient delivery is observed to several cell types, including endothelial, epithelial, fibroblasts and immune cells in the lungs. Hydrophobically modified siRNAs often induce strong immune responses after local delivery and thus can't be widely used. Surprisingly therapeutic distribution of Phosphorothioate enriched, fully modified siRNAs was observed after intratracheal administration. Delivery to relevant cell types was observed after administration of both monovalent and divalent compounds, with increased delivery after administration of divalent versus monovalent entities.

The presence of a two-thymidine linker between the conjugate and the siRNA does not impact siRNA tissue distribution profile (FIG. 19 ). Three different siRNA structural configurations were studied to evaluate the impact of the nature of the linker on distribution of DCA-conjugated siRNA in liver, kidney, spleen, lung, heart, muscle and fat 1-week after a single SC injection with 20 mg/kg (n=5-6 mice per group ±SD), as measured by a PNA hybridization assay. Data show efficient delivery of all three siRNA configurations to the lung.

The presence of a two-thymidine linker was found to increase DCA-conjugated siRNA silencing in multiple tissues, as measured by huntingtin and cyclophilin B mRNA expression (FIG. 20 ). Six mice per group were injected with siRNA by SC injection (FVB/N mice); 20 mg/kg; and tissues were collected one week after injection. The mRNA levels were measured using QuantiGene® (Affymetrix), and normalized to a housekeeping gene, Hprt (Hypoxanthine-guanine phosphoribosyl transferase), and presented as percent of PBS (Phosphate buffered saline) control (mean±SD).

Six siRNAs were then designed containing different numbers of 3′ exNA modifications and phosphorothioates. (FIG. 21 ) for lung delivery via systemic (SC) delivery. In FIG. 22 the impact of the chemical composition on siRNA distribution and efficacy is evaluated. The data show increased accumulation of DCA-conjugated siRNAs with exNA modifications compared to those without exNAs in all tissues including the lungs. Injections were done SC, 20 mg/kg, in three mice, for 1 week, and distributions were assessed in the PNA hybridization assay. Results show for the p2 scaffold show that 4PS-exNa accumulation was comparable to 7PS in liver, spleen, and lung; 4PS-exNa>7 PS in heart, muscle, fat; 7PS>4PS-exNa in kidney. p5 scaffold: 4PS-exNa>2PO-exNa 2PS-exNa≥4 PS>2 PS.

Increased silencing of DCA-conjugated siRNAs was achieved with exNA modifications compared to those without exNAs in all tissues including the lungs (FIG. 23 ). Target mRNA silencing (Htt) after systemic administration of siRNAs conjugated to DCA and containing different numbers of 3′ exNA modifications and phosphorothioates was assessed in various organ tissues including the liver, kidney, spleen, muscle, lung, heart, adrenal glands, and fat. The delivery was done by SC injection, 20 mg/kg, in five mice per group for one week, and quantification was done with the bDNA QuantiGene assay.

SiRNAs were then designed for lung delivery, to evaluate the impact of the chemical composition on siRNA distribution and efficacy (FIG. 24 ). The distribution and delivery throughout the lung of mono- and divalent siRNAs (Cy-3) is shown in FIG. 25A, and of DCA and EPA conjugated siRNAs (Cy-3) in FIG. 25B. Delivery via intratracheal injection (mono and divalent conjugates) were at 20 nmol for monovalent, and 40 nmol for divalent siRNAs, in two mice per group, and tissues were harvested after 24 h. Subcutaneous delivery (EPA and DCA conjugates) was at 40 nmol per construct in three mice per group and tissues were harvested after 48 h. Magnification is 5×, and the scale=1 mm.

Divalent siRNAs distribute to all cells of the lungs and saturate both alveolar and epithelial (club) cells 24 hours after intratracheal administration. Accumulation of mono and di-valent siRNA (Cy-3, red) after intratracheal administration is shown in FIG. 26 . Distribution is shown throughout the lung (FIG. 26A), in club cells (FIG. 26B; green), and in alveoli type II cells (FIG. 26C; green) as compared to PBS controls. FIG. 27 depict results obtained with EPA and DCA conjugates delivered by subcutaneous (SC) administration as assessed after 48 h.

Divalent siRNAs showed the highest amount of uptake among mon-divalent, EPA-conjugated and DCA-conjugated siRNA's, both in alveolar cells as well as in club cells (FIG. 28A and FIG. 28C, respectively, as quantitated in FIG. 28B. and FIG. 28D, respectively). Quantitation was performed using cy3 fluorescence signal intensity and colocalization with markers of different cell types, siRNA accumulation was quantified after systemic (SC) administration of EPA and DCA conjugated siRNAs, and intratracheal administration of mono and di-valent siRNAs. All siRNAs delivered to cells throughout the lung but to different extents.

Monovalent, divalent, EPA-conjugated and DCA-conjugated siRNA 1 siRNAs delivered to cells throughout the lung, but to a different extent. Cy-3 signals were quantitated in total cells, immune cells, endothelial cells, epithelial cells and fibroblasts, after systemic (SC) administration of EPA and DCA conjugated siRNAs (FIG. 29A-29C) and after intratracheal (IT) administration of mono- and divalent siRNAs (FIG. 30A-30C).

There is a clear increased accumulation of di-siRNA compared mono siRNA or DCA- or EPA-conjugated siRNA in the lungs. The distribution and accumulation of mono and di-siRNAs in various tissues were assessed after intratracheal injection, and for DCA- and EPA-conjugated siRNA after SC injection, as shown in FIG. 31 . Amounts injected by intratracheal administration were 7.5 and 15 nmol, for mono- and divalent siRNA, respectively, and 40 nmol for EPA/DCA conjugated siRNA, in groups of three mice each, followed by quantitation of siRNA accumulation after a week using the PNA hybridization assay.

A low dose of di-siRNA achieved the best silencing in lungs without silencing the gene in other tissues. FIG. 32A-32H and FIG. 33 show target mRNA silencing (Htt) after intratracheal administration of mono and di-siRNAs (7.5 or 15 nmol, respectively) in liver, kidney, spleen, lung, heart, adrenal glands; muscle and fat tissues, showing that divalent siRNA selectively silences the Htt mRNA in the lung.

Example 4. Additional SARS-CoV-2 Target Sites

Materials and Methods for Example 4

siRNA Treatment and Infection Assay

siRNAs were complexed with Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol and added to the wells of a 96-well plate at the desired final concentration (10 nM for screening, concentration range for dose response assays). To each of these wells was added A549 cells expressing the human ACE2 receptor at a final concentration of 15,000 cells/well. The plate was incubated for 36 hours at 37° C., 5% CO₂. After 36 hours of siRNA treatment, SARS CoV-2 virus was added to each well at a final MOI of 0.1 (unless otherwise specified) and incubated for 1 hour. One hour after virus addition, wells were washed with PBS and incubated for a further 48 hours followed by processing for molecular biology analysis.

Viral RNA Abundance

Viral RNA abundance was measured from the supernatants of cells 48 hours post virus infection. Briefly, 100 μL of cell culture supernatant from cells was treated with Trizol-LS according to the manufacturer's recommendations. RNA was then isolated using standard protocols using chloroform and ethanol. The abundance of SARS-CoV-2 Nucleocapsid RNA was measured by real-time quantitative PCR using the QuantiFast Pathogen RT-PCR kit (Qiagen) with the 2019-nCoV CDC qPCR Probe (IDT).

Viral Protein Abundance

Viral protein abundance was measured using immunofluorescence staining with anti-SARS-CoV-2 spike antibody to detect viral spike protein. Briefly, cells were fixed using 4% paraformaldehyde and serial ethanol dehydration followed by standard immunofixation procedures to detect proteins. Cells staining positive for the viral spike protein were counted using a fluorescence microscope.

Additional siRNAs and ASOs were tested against various SARS-CoV2 genes. siRNAs and ASOs were tested in A549-ACE2 cells and silencing was assessed using the psi-check reporter system. siRNA concentration: 10 nM; ASO concentration: 25 nM; Time point: 72 hours. As shown in FIG. 34 , numerous siRNAs and ASOs were cabable of reducing SARS-CoV-2 mRNA levels by 99%, including several that reduce levels to a similar level as remdesivir, an approved therapy for COVD-19.

Based on the results of the screen performed in FIG. 34 , several top hits were tested in dose response experiments. siRNAs 1a_2290, 7a_27751, 1ab_18571, and N_29293 were each tested at concentrations of 10 nM, 2 nM, 0.4 nM, and 0.08 nM. As shown in FIG. 35 , each of the tested siRNAs were able to effectly silence SARS-CoV-2 mRNA at several doses. Moreover, the tested siRNAs led to reduction of SARS-CoV-2 spike protein positive cells.

An additional dose response experiment was performed as described above for FIG. 35 . In this experiment, cells were infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.1 and 0.4. As shown in FIG. 36 , the tested siRNAs were able to effectly silence SARS-CoV-2 mRNA at several doses and at both MOIs tested.

An additional screen of siRNAs was performed targeting the orf7a SARS-CoV2 gene. siRNAs were tested in in A549-ACE2 cells and the data reported was relative mRNA abundance of the targeted orf7a SARS-CoV2 gene and the percent of cells that are positive for the SARS-CoV2 spike protein. siRNA concentration: 10 nM; Time point: 72 hours. As shown in FIG. 37 , numerous siRNAs were identified that effectively target orf7a.

The antisense and sense strands of the siRNAs tested in FIGS. 35 and 36 are shown below in Table 14.

TABLE 14 Antisense and Sense sequences of select siRNAs. Orf7a_27751 SEQ ID NO: Antisense 5′ UGAAAGUUCAAUCAUUCUUUU 3′ 2575 Sense 5′ AAUGAUUGAACUUUCA 3′ 2576 N_29293 Antisense 5′ UGAAAUUUGGAUCUUUGUUUU 3′ 2577 Sense 5′ AAAGAUCCAAAUUUCA 3′ 2578 Orf1a_2290 Antisense 5′ UAAGCUUAAAGAAUGUCUUUU 3′ 2579 Sense 5′ ACAUUCUUUAAGCUUA 3′ 2580 Orf1ab_18571 Antisense 5′ UCAAAUACGACUCUGUCUUUU 3′ 2581 Sense 5′ ACAGAGUCGUAUUUGA 3′ 2582

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references, patents, patent applications, and websites) that maybe cited throughout this application are hereby expressly incorporated by reference in their entirety for any purpose, as are the references cited therein. The disclosure will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology and cell biology, which are well known in the art.

The present disclosure also incorporates by reference in their entirety techniques well known in the field of molecular biology and drug delivery. These techniques include, but are not limited to, techniques described in the following publications:

Atwell et al. J. Mol. Biol. 1997, 270: 26-35;

Ausubel et al. (eds.), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &Sons, NY (1993);

Ausubel, F. M. et al. eds., SHORT PROTOCOLS IN MOLECULAR BIOLOGY (4th Ed. 1999) John Wiley & Sons, NY. (ISBN 0-471-32938-X);

CONTROLLED DRUG BIOAVAILABILITY, DRUG PRODUCT DESIGN AND PERFORMANCE, Smolen and Ball (eds.), Wiley, New York (1984);

Giege, R. and Ducruix, A. Barrett, CRYSTALLIZATION OF NUCLEIC ACIDS AND PROTEINS, a Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press, New York, N.Y., (1999);

Goodson, in MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, pp. 115-138 (1984);

Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981;

Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);

Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST (National Institutes of Health, Bethesda, Md. (1987) and (1991);

Kabat, E. A., et al. (1991) SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242;

Kontermann and Dubel eds., ANTIBODY ENGINEERING (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5).

Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990);

Lu and Weiner eds., CLONING AND EXPRESSION VECTORS FOR GENE FUNCTION ANALYSIS (2001) BioTechniques Press. Westborough, Mass. 298 pp. (ISBN 1-881299-21-X).

MEDICAL APPLICATIONS OF CONTROLLED RELEASE, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974);

Old, R. W. & S. B. Primrose, PRINCIPLES OF GENE MANIPULATION: AN INTRODUCTION TO GENETIC ENGINEERING (3d Ed. 1985) Blackwell Scientific Publications, Boston. Studies in Microbiology; V.2:409 pp. (ISBN 0-632-01318-4).

Sambrook, J. et al. eds., MOLECULAR CLONING: A LABORATORY MANUAL (2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN 0-87969-309-6).

SUSTAINED AND CONTROLLED RELEASE DRUG DELIVERY SYSTEMS, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978

Winnacker, E. L. FROM GENES TO CLONES: INTRODUCTION TO GENE TECHNOLOGY (1987) VCH Publishers, NY (translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).

EQUIVALENTS

The disclosure may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting of the disclosure. Scope of the disclosure is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are therefore intended to be embraced herein. 

1. An oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of SEQ ID NO:
 1. 2. The oligonucleotide compound of claim 1, comprising: a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 2-10, a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 20-nucleotide targets in Table 6A; complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; comprising no more than 3 mismatches with the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10; or comprising full complementarity to the SARS-CoV-2 nucleic acid sequence of any one of SEQ ID NOs: 1-10. 3-7. (canceled)
 8. An oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to an Angiotensin I Converting Enzyme 2 (ACE2) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 11D or Table 12C.
 9. The oligonucleotide compound of claim 8, comprising a sequence substantially complementary to an ACE2 nucleic acid sequence of any one of the 45-nucleotide target gene regions of ACE2_119, ACE2_336, ACE2_349, ACE_1034, ACE_1775, ACE_784, ACE_908, and ACE_1071, as recited in Table 12C
 10. An oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a FURIN nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 1 IC or Table 12D.
 11. The oligonucleotide compound of claim 10, comprising a sequence substantially complementary to a FURIN nucleic acid sequence of any one of the 45-nucleotide target gene regions of FURIN_443, FURIN_1959, FURIN_2711, FURIN_2712, FURIN 3524, and FURIN 3526, as recited in Table 12D.
 12. An oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to an Interleukin 6 (IL-6) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 11B or Table 12B.
 13. An oligonucleotide compound comprising 15 to 35 bases in length, comprising a sequence substantially complementary to a Transmembrane Serine Protease 2 (TMPRSS2) nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 11A or Table 12A.
 14. (canceled)
 15. The oligonucleotide compound of claim 1, wherein the oligonucleotide compound comprises one or more modified nucleotide, optionally wherein: the oligonucleotide compound comprises at least 80% chemically modified nucleotides or 100% chemically modified nucleotides; and/or the one or more modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof, optionally wherein: each modification of the ribose group is independently selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH₂, and a constrained nucleotide, optionally wherein the constrained nucleotide is selected from the group consisting of a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA^(NC)), an alpha-L-locked nucleic acid, a tricyclo-DNA, and any combination thereof; each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N⁶-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups; and/or each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, and phosphotriester modification, optionally comprising 4-16 phosphorothioate modifications or 6-13 phosphorothioate modifications. 16-24. (canceled)
 25. The oligonucleotide compound of claim 1, wherein the oligonucleotide compound comprises at least one modified internucleotide linkage of Formula I:

wherein: B is a base pairing moiety; W is selected from the group consisting of O, OCH₂, OCH, CH₂, and CH; X is selected from the group consisting of halo, hydroxy, and C₁₋₆ alkoxy; Y is selected from the group consisting of O—, OH, OR, NH—, NH₂, S—, and SH; Z is selected from the group consisting of O and CH₂; R is a protecting group; and

is an optional double bond. 26-27. (canceled)
 28. The oligonucleotide compound of claim 1, wherein the oligonucleotide compound comprises an antisense oligonucleotide or a double stranded (ds) RNA, optionally wherein the dsRNA comprises an antisense strand and a sense strand, optionally wherein: the antisense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length; the sense strand comprises about 15 nucleotides to 25 nucleotides in length, optionally 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length; the dsRNA comprises a double-stranded region of 15 base pairs to 20 base pairs, optionally 15 base pairs, 16 base pairs, 18 base pairs, or 20 base pairs; the dsRNA comprises a blunt-end; the dsRNA comprises at least one single stranded nucleotide overhang; the dsRNA comprises about a 2-nucleotide to 5-nucleotide single stranded nucleotide overhang; the dsRNA comprises at least 70% 2′-O-methyl nucleotide modifications; the antisense strand comprises at least 70% 2′-O-methyl nucleotide modifications or about 70% to 90% 2′-O-methyl nucleotide modifications; the sense strand comprises at least 65% 2′-O-methyl nucleotide modifications or 100% 2′-O-methyl nucleotide modifications; the sense strand comprises one or more nucleotide mismatches between the antisense strand and the sense strand, optionally wherein the one or more nucleotide mismatches are present at positions 2, 6, and 12 from the 5′ end of sense strand; the antisense strand comprises a 5′ phosphate, a 5′-alkyl phosphonate, a 5′ alkylene phosphonate, or a 5′ alkenyl phosphonate; the antisense strand comprises a 5′ vinyl phosphonate; the nucleotides at positions 1 and 2 from the 5′ end of antisense strand are connected to adjacent ribonucleotides via phosphorothioate linkages; and/or (A) the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; (3) the nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are not 2′-methoxy-ribonucleotides; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides; and (7) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; or (B) the dsRNA comprises an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not a 2′-methoxy-ribonucleotide; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 6, 7, 8 and 10 from the 5′ end of the sense strand are not a 2′-methoxy-ribonucleotide; and (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages. 29-51. (canceled)
 52. The dsRNA of claim 28, wherein the antisense strand, the sense strand, or both the antisense and sense strand comprises 1 to 5 internucleotide linkages of Formula I, optionally wherein: the 3′ end of the antisense strand comprises 1 to 5 internucleotide linkages of Formula I; the 3′ end of the antisense strand comprises 4 consecutive internucleotide linkages of Formula I; or the 3′ end of the antisense strand comprises 4 consecutive internucleotide linkages of Formula I. 53-55. (canceled)
 56. The dsRNA of claim 28, wherein a functional moiety is linked to one or both of the 5′ end and 3′ end of the sense strand, optionally wherein: the functional moiety is linked to one or both of the antisense strand and sense strand by a linker, optionally wherein the linker comprises a divalent or trivalent linker, optionally selected from the group consisting of:

wherein n is 1, 2, 3, 4, or 5; the linker comprises an ethylene glycol chain, an alkyl chain, a peptide, an RNA, a DNA, a phosphodiester, a phosphorothioate, a phosphoramidate, an amide, a carbamate, or a combination thereof; the linker comprises a dTdT dinucleotide; the functional moiety is linked to the 3′ end of the sense strand by a dTdT dinucleotide followed by the linker

wherein n is 1; and/or the trivalent linker further links a phosphodiester or phosphodiester derivative, optionally wherein the phosphodiester or phosphodiester derivative is selected from the group consisting of:

wherein X is O, S or BH₃; and/or the functional moiety comprises a hydrophobic moiety, optionally wherein: the hydrophobic moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides, nucleoside analogs, endocannabinoids, vitamins, and a mixture thereof, optionally wherein: the steroid selected from the group consisting of cholesterol and Lithocholic acid (LCA): the fatty acid selected from the group consisting of Eicosapentaenoic acid (EPA), Docosahexaenoic acid (DHA) and Docosanoic acid (DCA); or the vitamin is selected from the group consisting of choline, vitamin A, vitamin E, retinoic acid, alpha-tocopheryl succinate, and derivatives or metabolites thereof. 57-72. (canceled)
 73. A double stranded (ds) RNA, comprising an antisense strand and a sense strand, each strand with a 5′ end and a 3′ end, wherein: (1) the antisense strand comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; (2) the antisense strand comprises at least 70% 2′-O-methyl modifications; (3) the nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand are not a 2′-methoxy-ribonucleotide; (4) the nucleotides at positions 1-2 to 1-7 from the 3′ end of the antisense strand are connected to each other via phosphorothioate internucleotide linkages; (5) a portion of the antisense strand is complementary to a portion of the sense strand; (6) the sense strand comprises at least 70% 2′-O-methyl modifications; (7) the nucleotides at positions 6, 7, 8 and 10 from the 5′ end of the sense strand are not a 2′-methoxy-ribonucleotide; (8) the nucleotides at positions 1-2 from the 5′ end of the sense strand are connected to each other via phosphorothioate internucleotide linkages; and (9) the antisense strand comprises at least one modified intersubunit linkages of Formula II:

wherein: B is a base pairing moiety; W is O or O(CH₂)_(n) ¹, wherein n¹ is 1 to 10; X is selected from the group consisting of H, OH, OR¹, F, SH, SR, NR² ₂ and C₁₋₆-alkoxy; Y is selected from the group consisting of O—, OH, OR, OR², NH—, NH₂, NR² ₂, BH₃, S—, R¹, and SH; Z is O or O(CH₂)_(n) ² wherein n² is 1 to 10; R¹ is alkyl, allyl or aryl; and R² is alkyl, allyl or aryl.
 74. The dsRNA of claim 73, wherein: the 3′ end of the antisense strand comprises four consecutive modified intersubunit linkages of Formula II; the antisense strand comprises a 5′ vinyl phosphonate; the antisense strand is 20 nucleotides in length, 21 nucleotides in length, or 22 nucleotides in length; the sense strand is 15 nucleotides in length, 16 nucleotides in length, 18 nucleotides in length, or 20 nucleotides in length; and/or a functional moiety is linked to the 3′ end of the sense strand by a linker, optionally wherein: the functional moiety comprises Eicosapentaenoic acid (EPA) or Docosanoic acid (DCA); the linker comprises a dTdT dinucleotide; and/or the functional moiety is linked to the 3′ end of the sense strand by a dTdT dinucleotide followed by the linker

wherein n is
 1. 75-82. (canceled)
 83. A combination comprising two or more oligonucleotide compounds of claim 1, wherein at least two oligonucleotides or dsRNA comprise complementarity to a different SARS-CoV-2 nucleic acid sequence, optionally comprising two, three, four, or five oligonucleotide compounds or dsRNA, each comprising complementarity to a different SARS-CoV-2 nucleic acid sequence. 84-85. (canceled)
 86. The combination of claim 83, comprising a first oligonucleotide compound, a second oligonucleotide compound, and a third oligonucleotide compound, wherein: i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_8744, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_8744, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region N_29293, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region N_29293, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26470, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27123, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23174, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27032, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_416, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26305, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27565, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orfla_9679, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region E_26369, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27656, as recited in Table 6A; i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region orflab_21391, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region M_27032, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; or i) the first oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 3a_25868, as recited in Table 6A; ii) the second oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region 7a_27751, as recited in Table 6A; and iii) the third oligonucleotide compound comprises a sequence substantially complementary to the 45-nucleotide target gene region S_23774, as recited in Table 6A.
 87. A combination comprising one or more oligonucleotide compounds for inhibiting the expression of a SARS-CoV-2 gene and one or more oligonucleotide compounds for inhibiting the expression of one or more of an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and a IL-6R gene.
 88. A pharmaceutical composition for inhibiting the expression of one or more of a SARS-CoV-2 gene, an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and a IL-6R gene in a cell of an organism, comprising the oligonucleotide compound of claim 1 and a pharmaceutically acceptable carrier, optionally wherein the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 genes, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 50% or by at least 75%. 89-94. (canceled)
 95. A method for inhibiting expression of one or more of a SARS-CoV-2 gene, an ACE2 gene, a FURIN gene, an IL-6 gene, a TMPRSS2 gene, and an IL-6R gene in a cell, the method comprising: (a) introducing into the cell an oligonucleotide compound of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene thereby inhibiting expression of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene in the cell.
 96. A method of treating or managing a SARS-CoV-2 infection, comprising administering to a patient in need of such treatment a therapeutically effective amount of the oligonucleotide compound of claim 1, optionally wherein: the oligonucleotide compound is administered by intratracheal (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof; the oligonucleotide compound is administered sequentially or simultaneously; administering the oligonucleotide compound causes a decrease in one or more of SARS-CoV-2 gene mRNA, ACE2 gene mRNA, FURIN gene mRNA, IL-6 gene mRNA, TMPRSS2 gene mRNA, and IL-6R gene mRNA in the lung; and/or the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 50% or at least 75%. 97-101. (canceled)
 102. A vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes an oligonucleotide compound of claim 1, optionally wherein the oligonucleotide compound inhibits the expression of one or more of the SARS-CoV-2 gene, the ACE2 gene, the FURIN gene, the IL-6 gene, the TMPRSS2 gene, and the IL-6R gene by at least 30%, at least 50%, or at least 75%. 103-105. (canceled)
 106. A cell comprising the vector of claim
 102. 107. A recombinant adeno-associated virus (rAAV) comprising the vector of claim 102 and an AAV capsid.
 108. A branched oligonucleotide compound comprising two or more of the oligonucleotide compounds or dsRNA of claim 1 covalently bound to one another, optionally wherein the oligonucleotide compounds are covalently bound to one another by way of a linker, spacer, branching point, or a combination thereof.
 109. (canceled)
 110. A method of treating or managing a SARS-CoV-2 infection, comprising administering to a patient in need of such treatment a therapeutically effective amount of the branched oligonucleotide compound of claim 108, optionally wherein: the branched oligonucleotide compound is administered by intratracheal (IT) injection, intravenous (IV) injection, subcutaneous (SQ) injection, or a combination thereof; and or the branched oligonucleotide compound accumulates in lung tissue to a greater extent than a non-branched oligonucleotide compound when administered by intratracheal (IT) injection. 111-112. (canceled)
 113. A branched RNA compound comprising: two or more RNA molecules each comprising 15 to 35 nucleotides in length, and a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence, wherein the two or more RNA molecules are connected to one another by one or more moieties independently selected from a linker, a spacer, a branching point, and a combination thereof.
 114. The branched RNA compound of claim 113, comprising a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A, optionally wherein: each RNA molecule comprises 15 to 25 nucleotides in length; each RNA molecule comprises a dsRNA comprising a sense strand and an antisense strand, wherein each antisense strand independently comprises a sequence substantially complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A, optionally wherein the antisense strand comprises a portion having the nucleic acid sequence of any one of the antisense strands recited in Table 6B; each RNA molecule comprises complementarity to at least 10, 11, 12 or 13 contiguous nucleotides of a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; each RNA molecule comprises no more than 3 mismatches with a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A; and/or each RNA molecule comprises full complementary to a SARS-CoV-2 nucleic acid sequence of any one of the 45-nucleotide target gene regions in Table 6A. 115-198. (canceled)
 199. A method of delivering an oligonucleotide compound to the lung of a patient, comprising administering the oligonucleotide compound, wherein the oligonucleotide compound is conjugated to a functional moiety selected from Eicosapentaenoic acid (EPA) and Docosanoic acid (DCA). 200-207. (canceled) 